Lab #3 - Membrane Transport

Lab #3 - Membrane Transport Lecture Notes

In today’s experiments we will explore membrane transport processes, focusing on passive transport, specifically diffusion of molecules through various types of matter and across semipermeable membranes

1. Lab Manual, Ch 5, Ex 5-1- Diffusion

Diffusion - the movement of molecules/atoms from an area of high density/concentration to an area of low density/concentration

all particles have kinetic energy; they vibrate and move randomly, in what is known as Brownian Motion - as particles move, they bump into each other and disperse, naturally moving into areas of lower concentration, until eventually there is no net diffusion occurring - this is known as equilibrium

the rate of diffusion can depend on several factors; one of these factors is the state of matter that the particle is in - instructor’s demonstration

Questions: diffusion is faster in what type of matter - gas, liquid or solid? Which state of matter has the highest kinetic energy? Which state has the most space between the particles?
During the instructor’s demonstration - keep in mind, what is the difference in time scale (seconds, minutes, hours?) for diffusion in a gas vs. liquid vs. solid?
Diffusion also depends on temperature - instructor’s demonstration - hypothesize - in which solution will the dye move faster? Why? Which solution has the greater kinetic energy?
Diffusion can also depend on the size or molecular weight of the particle - looking at the agarose plate, you can come up with a hypothesis about which particle has the highest molecular weight, and which the lowest, based on rates of diffusion - was your hypothesis correct? If not, what other characteristics of the molecules may have influenced their rates of diffusion?
Rates of diffusion also depend on the steepness of the concentration gradient - what we will observe during the rate of osmosis experiment
Diffusion is a passive process - does not require energy; therefore, particles can only move down their concentration gradients (from a region of high concentration to a region of low concentration); diffusion cannot occur against the concentration gradient of the diffusing particle
In contrast, active transport requires energy in the form of ATP - molecules can be actively transported against their concentration gradients (eg., the Na/K ATPase)

Cell membranes allow the diffusion of molecules into/out of the cell, but these membranes are selectively permeable - only some substances are allowed through the membrane. Whether a particle is allowed through depends on its size, charge, polarity and hydrophobicity:

Small, nonpolar molecules like oxygen, carbon dioxide can diffuse directly through the cell membrane
Molecules that are similar in structure to the cell membrane can also pass directly through, eg., steroid hormones
Molecules that are polar (water) or carry a charge (ions) must pass through channels that are selective for specific molecules
Other molecules diffuse through the cell membrane via carrier molecules (proteins and sugars)
In today’s experiments, the dialysis tubing you will use is made of a cellulose membrane that allows the diffusion of molecules according to size only (molecules less than/equal to 14000 daltons can diffuse through)

2. Lab Manual Ch 5 Ex 5-4 - Dialysis

In the dialysis experiment, you will be observing the ability of the dialysis membrane to sort molecules based on size; you should be able to come up with a hypothesis about which molecules will be able to diffuse out of the dialysis tubing, and which will not - will any of the molecules be found both inside and outside the dialysis bag?
glucose = small monosaccharide
sucrose = disaccharide - 2 sugar molecules linked together
starch = polysaccharide - many sugar molecules linked together
water = small, polar but non-charge molecule

3. Lab Manual Ch 5 Ex 5-2 - Osmosis and Volume Changes in Cells, and Ex 5-3 -

Rate of Osmosis

Osmosis - diffusion of water through a semipermeable membrane

The movement of water across cell membranes can affect cell volume, shape and cell survival.

Isotonic solutions = 290-300 mosm = 0.9% saline = no net diffusion of water

Hypertonic solutions = > 300 mosm = net diffusion of water into solution, out of cells

Hypotonic solutions = < 300 mosm = net diffusion of water out of solution, into cells

In Ex 5-2, you will observe what happens to rat red blood cells when they are placed in hypertonic, isotonic or hypotonic solutions - you should be able to think of a hypothesis to predict what will happen to the red blood cells in each of these solutions. In Ex 5-3, you will observe how the rate at which water moves across the dialysis membrane is affected by the concentration of solutes on either side of the membrane.

PROCEDURE SHEET FOR MEMBRANE TRANSPORT LAB

A FEW HINTS ABOUT THE FOLLOWING EXPERIMENTS:
1. Set up experiment 2 first and get it going - it takes the longest and is the most complicated.
2. Divide labor among the members of your lab group - at least 2 people should be working on experiment 2.
3. Experiment 3 can be set up and done last - it only takes about 15 minutes.
4. Be sure to read through ALL the instructions before beginning any of the experiments!

Experiment 1: Osmotic Changes in Red Blood Cells
The movement of water across a semi-permeable membrane is given a special name, osmosis. The movement of water across the cell membrane is of utmost importance to all the cells in the body, because it can affect cell volume, cell shape and ultimately, cell survival. In this experiment you will change the rate and direction of water movement by osmosis, using different extracellular solutions. You will observe the effect these osmotic changes have on cell volume and shape. These solutions can be described using terms that describe the solute concentration of the solutions relative to the solute concentration inside the red blood cells:

Hypertonic: It has a higher solute concentration than the cell. Water will diffuse out of
the cell and into the solution, causing the cell to shrink (crenation).

Hypotonic: It has a lower solute concentration than the cell. Water will diffuse out of
the solution and into the cell, causing the cell to swell and possibly burst (lysis)

Isotonic: It has the same solute concentration as the cell. There will be no net
movement of water, and the cell will neither shrink nor swell.

Remember that these terms are relative - a solution with a 10% solute concentration will be hypertonic to one with a 5% solute concentration. However, the 10% solution is hypotonic to a solution with a 15% solute concentration.

MATERIALS:
compound microscope
1-2 microscope slides and cover slips
3 solutions: 0.9% NaCl, distilled water, 10 % NaCl solution
diluted rat blood

1. Put a drop of diluted rat blood on a slide, add one drop of isotonic saline, and drop a cover slip onto the slide. Observe the RBCs using the high dry objective (43-45X). Make a drawing or write a description of the cells’ size and shape in the space provided on the next page.

2. Place a drop of 10% NaCl at one edge of the cover slip and wick it through (place a piece of Kimwipe at the other edge of the cover slip to draw the solution under the cover slip). Wait a few minutes, then observe the size and shape of the cells. Note any differences in the space on the next page.

3. Place a drop of distilled water at one edge of the cover slip and wick it through. Note the size and shape of the cells after a few minutes.

4. Alternative method: Follow step 1; then, get a fresh slide and 2 more cover slips.

Put a drop of rat blood at one end of the slide, and add a drop of 10% NaCl to the blood, and put on a cover slip. At the other end of the slide, place another drop of rat blood, add a drop of distilled water, and a cover slip. Compare the size and shape of the cells at each end of the slide under the microscope using the high dry objective (43-45X).

5. For each of the solutions you applied to the red blood cells, describe: 1) What happened to the shape and size of the cells; 2) Whether the solution you applied was isotonic, hypertonic, or hypotonic to the cells; 3) The net direction of water movement (into the cells, out of the cells, no net movement).

10 % NaCl solution:

distilled water:

0.9 % NaCl:

Experiment 2: Rate of Osmosis
In experiment 1 you looked at the effect of water movement on the size and shape of cells. In this experiment you will examine the effect of a concentration gradient on the speed of water movement across a semipermeable membrane (dialysis tubing). You will compare the rate of osmosis for 3 different combinations of solutions:

Bag Setup
BAG INSIDE BAG IN BEAKER
1 tap water 20% sucrose

2 1% sucrose tap water

3 10% sucrose tap water

MATERIALS:
dialysis bags soaking in water
3 beakers, 1 funnel
rubber bands
solutions: 10% sucrose, 20% sucrose, 1% sucrose
paper towels; watch

NOTE: Follow the procedure for each dialysis bag until completion before starting another one - this experiment requires a sequence of timed measurements - don’t try to prepare all the dialysis bags simultaneously!

1. Take one dialysis bag out of the beaker and tie off one end (instructor will demonstrate how to tie off the bags to prevent leaks). Fill the bag with 20 mls of tap water, using the funnel. Squeeze any air out of the bag, being careful NOT to use your fingertips (the oil on the skin of your fingertips can damage the dialysis membrane). Tie off the opposite end of the bag.

2. Dry the bag thoroughly on paper towels, especially the knotted ends. Weigh the bag on the balance

3. Put the bag in a labeled 400 ml beaker, and fill the beaker with 20% sucrose to just cover the bag - NOTE THE TIME.

4. Fill the second dialysis bag with 1% sucrose, tie it off, dry it, weigh it, put it in a separate, labeled, 400 ml beaker with enough tap water to cover the bag, and again NOTE THE TIME.

5. Fill the third dialysis bag with 10% sucrose, tie it off, dry it, weigh it, put it in a separate, labeled 400 ml beaker with enough tap water to cover the bag, and once more NOTE THE TIME.

6. Weigh each bag every 15 minutes for one hour - make sure you dry the bag thoroughly before each weighing. Also, make sure the bags stay submerged in the liquid - if necessary, weight them down with a pen or pencil.

7. You may use the chart below to keep track of your weighing times and the weights of the dialysis bags.

8. Graph the weight change of each bag as a function of time for each experiment (due next class period as part of your Lab Report).

EXPERIMENTAL RESULTS

	Weight at T=0	Weight at T = 15 min	Weight at T =30min	Weight at T = 45 min	Weight at T = 60 min
Bag 1
Bag 2
Bag 3

CALCULATIONS
1. Initial rate of osmosis = weight at 15 min - weight at 0 min / 15 min

You will calculate the initial rates of osmosis for bags 1, 2 and 3 as part of your lab report, due next lab session.

2. Given the formula for the initial rate of osmosis, write the formula for the final rate of osmosis below:

You will calculate the final rates of osmosis for bags 1, 2 and 3 as part of your lab report, due next lab session.

THOUGHT QUESTIONS
Why did some of the dialysis bags gain weight while other bags lost weight? What produced the difference in the rate of weight change among the 3 bags? Do you think there will be a difference in the initial and final rates of osmosis for any of the bags? Why or why not? What molecule was moving across the dialysis membrane to produce the weight changes observed in the dialysis bags?

Experiment 3: Dialysis
The ability of a molecule to diffuse through a semipermeable membrane depends on its size and shape. The process of dialysis takes advantage of a molecule’s ability to diffuse across a semipermeable membrane in order to separate large and small molecules. In this experiment you will compare the ability of glucose and starch molecules to cross dialysis tubing, a semipermeable membrane. The dialysis tubing we are using allows the passage of molecules smaller than 14000 daltons. While you are doing this experiment keep in mind that glucose is a monomer (a single sugar molecule) and starch is a polymer made up of several sugar molecules linked together.

MATERIALS:
1 piece of dialysis tubing, soaking in water
beaker
funnel
4 test tubes
test tube holder
colored tape and marking pen
iodine solution and Benedict’s solution
starch (10%) and glucose (5%) solution
rubber bands

1. Tie off one end of the dialysis tubing with rubber bands , as you did in experiment 2

2. Using a funnel, fill the bag with ~20 mls of the starch/glucose solution. Make sure all the air is out of the bag, and tie off the other end with twine.

3. Immerse the bag in a beaker of tap water, and make sure the bag stays under the surface of the water.

4. Let the bag sit in the beaker of water for 15 minutes.

5. Label 4 test tubes:
    IN - starch
    OUT - starch
    IN - glucose
    OUT - glucose

6. At the end of 15 minutes, cut one end off the dialysis bag and pour a few mls (doesn’t matter how many exactly) into the "IN" test tubes. Pour a few mls of the beaker water into the "OUT" test tubes.

7. Add 10 drops of iodine solution to the tubes labeled: IN - starch & OUT - starch
A dark blue color indicates the presence of starch. Record your results in the table below.

8. Add 10 drops of Benedict’s solution to the tubes labeled: IN - glucose & OUT - glucose
Put the test tubes containing the Benedict’s solution in a boiling water bath (on the side bench) for 1-2 minutes. The blue color will change to green, orange or yellow in the presence of glucose. Record your results in the table below.

THOUGHT QUESTIONS
Based on what you know about the relative size of glucose and starch molecules, you should be able to predict which molecule(s) will diffuse out of the bag and which molecule(s) will stay inside the bag.

EXPERIMENT RESULTS

Test Tube	Presence of Starch*	Presence of Glucose*
IN - starch		----------------------------
OUT - starch		----------------------------
IN - glucose	---------------------------
OUT - glucose	---------------------------

* indicate absence of molecule with a "-" and presence of molecule with a "+"