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Lab 6 Passive Transport (Team 4)

Page history last edited by Karen Huang 10 years, 6 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

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. 

  

 

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. 

 

The plasma membrane is composed of a phospholipid bilayer. The bilayer acts as a barrier to selectively allow certain substances in and keep others out. The heads of the phospholipids are hydrophilic which means they attract to water, while the tails are hydrophobic which allows for the bilayer to be formed. Kinks formed by double bonds in the hydrophobic tails allow the membrane to exist in a fluid state. The plasma membrane is also referred to as a mosaic model due to the various assortments of organic molecules that exist among the phospholipids. Cholesterols, proteins, and carbohydrates also assist in the function of the membrane. Integral proteins help transport substances that cannot pass through the bilayer or are unable to pass with enough speed and efficiency. Cholesterol helps to maintain membrane integrity; it does this because of its structure. The hydroxyl group aligns with the phosphate heads while the part of the steroid is attracted to the lipids and it creates less mobilization narrowing down the amount of substances allowed through only allowing smaller molecules to pass. Carbohydrates are eternal and attach to proteins. They bind cells together and are basically all together attachment sites of chemical messengers.

 

http://www.freethought-forum.com/images/anatomy2/phospholipid.gif

 

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.  

 

Osmosis is the movement of water across a selectively permeable membrane into a region of higher solute concentration. The water molecules continue to move across until equilibrium is reached and both sides have an equal distribution of solute and water. Osmosis is an example of passive transport and therefore does not require energy, this makes osmosis spontaneous.  A solution is isotonic if the concentration of dissolved solutes is equal on both sides of the gradient. If the concentration of the solutes is higher on the outside of the gradient than the inside, then the solution is hypertonic. If placed in a hypertonic solution, a cell will crenate or shrivel up since the water from the inside will rush to the outside so the solute concentrations are equal on both sides.  If the concentration of solutes is lower on the outside of the gradient than inside, then the solution is hypotonic. If placed in a hypotonic solution, a cell will lyse since the water from the outside will enter the cell to balance out the solute concentration. 

(Image from http://bio1903.nicerweb.com/Locked/media/ch07/07_12Osmosis-L.jpg)

 

File:Osmotic pressure on blood cells diagram.svg

 

(Image from http://upload.wikimedia.org/wikipedia/commons/7/76/Osmotic_pressure_on_blood_cells_diagram.svg)

 

 

 

 

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.  This 15mm dialysis tubing has small pores that allows only for the passage of water and not solutes.  Dialysis clips 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. Click on this link if your video won't load.

 

 

Materials

15 mm dialysis tubing (anywhere from 3-6 tubes are available) that hold 10mL of sucrose solution

30% stock solution of sucrose

 

400 mL beakers containing DI-water

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?
  3. You will be responsible for any dilutions of your 30% stock.  Think about how many different concentrations you want to test.  What is the final volume of your dilutions?
  4. In what units will you measure the rate of water movement? 
  5. What is the density of water?
  6. How long will you allow for the experiment to take place? 

Experimental Design: 

The purpose of this experiment is to investigate how concentration gradient influences the direction of net water flow during osmosis. To do this we will test different concentrations and compare results to see if concentration gradients influence the direction of net water flow during osmosis. The sucrose solution is a 30% stock solution so to keep things simple, the 4 concentrations we will test will be 30%, 20%, 10%, and 0%. Since the dialysis tubing only holds 10 mL of sucrose solution, we will use that number (it will also make things simpler during conversions). 

The amount of solution needed for 30% sucrose solution- 10mL                          The amount of DI water needed- 0mL

The amount of solution needed for 20% sucrose solution- 6.66 mL                      The amount of DI water needed- 3.33 mL

The amount of solution needed for 10% sucrose solution- 3.33 mL                       The amount of DI water needed- 6.66 mL

The amount of solution needed for 0% sucrose solution- 0mL                               The amount of DI water needed - 10 mL

The rate of water movement will be measured in mL/min and the total amount of time that the experiment will take is 60 mins.

Density= mass/volume. The density of water is 1.

 

1. Prepare dialysis tubing with 4 separate concentrations of sucrose solutions (from the table above) using the video as a reference

2. Weigh the dialysis tubing. Record results

3. Place dialysis tubing in 200 mL of DI water for 60 mins.     

4. The tubing will be monitored at regular intervals of 15-20 mins.

5. After 60 mins, the tubing will be weighed again. Record results.

6. Compare the results from the original weight to the final weight.

7. Record the difference and put into to mL/min format.

 


Results:

 

 


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.  

 

 

 

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.


Results:

 

 

 

 


F.  Effect of Osmosis on Cells

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?

In the dH2O, water would move from the beaker into the cell (causing a hypotonic reaction) because the cell would have a higher concentration of solutes.

In the 0.9% NaCl, water would move freely in both direction since the solute concentration in the cell and in the cellular environment would be roughly the same (isotonic reaction).

In the 10% NaCl, water would move from the cell into the beaker (causing a hypertonic reaction) because the cellular environment would have a greater concentration of solutes and thus water would move out of the cell in an attempt to reach equilibrium. 

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

In a hypotonic reaction, the cell would overfill with water and lyse.

In a isotonic reaction, the cell would maintain its concentration, and thus maintain the same shape.

In a hypertonic reaction, the cell would lose water and crenate.

 

 

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.  Take video of each specimen and narrate what is happening in each environment.

     
IMAGE  #1
IMAGE  #2
IMAGE  #3

Presentation:

Record two presentations using the previous format (Introduction, Experimental Design/Execution, Results and Conclusions):

     1.  The diffusion of solutes across a permeable membrane (Section E).

     2.  Osmosis and its effects on cells (Sections D and F).

 

 

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