DIFFUSION, OSMOSIS, AND FILTRATION I.

INTRODUCTION As a Biology 204 student, at this stage you have already developed a good understanding of the concept of diffusion. Your grasp of diffusion has allowed you to see, for example, why oxygen enters the blood at the lungs while at the same time carbon dioxide leaves the blood at the lungs. In a similar way, in order to comprehend three major topics soon to come, a hands-on understanding of filtration and osmosis is key. These three topics are capillary dynamics, renal dynamics, and water balance. Capillary dynamics refers to the movement of clear fluid out of a blood capillary, where it is renamed “interstitial fluid.” This fluid bathes and nourishes the cells nearby. Most of this fluid is then returned into the capillary. If this process does not occur properly, a common but serious clinical problem, edema, occurs. Laypersons call minor edema “swelling” or “water retention,” and the understanding of how and why it occurs depends on the understanding of filtration and osmosis. Renal dynamics involves the production of urine by the blood. Its first step is filtration of clear fluid from the blood into kidney tubules, which depends on processes which we will examine in the lab today. Its second step is reabsorption of nutrients and water back into the blood. Most nutrient molecules move back into the blood by active transport, but water follows these molecules by the process of osmosis. Water balance is the topic that refers to the acquisition and loss of water, and its movement into and out of cells. Water is never actively transported within the body; instead, it follows other molecules by osmosis. What’s the link in all this to diffusion? As we will see, osmosis is the diffusion of water, under certain specific conditions. Refer to textbook pages 66-70 [66-69] and to Fig. 3.13, 3.16, and 3.17 [3.16 and 3.17] to develop your understanding of these topics.

II.

BASIC DEFINITIONS A.

Solution 1.

Solute

2.

Solvent 50

51 B.

C.

Diffusion 1.

Def.: Movement of molecules from an area higher concentration to an area of lesser concentration

2.

Driving force:

Osmosis 1.

Def.: Diffusion of water through a selectively permeable membrane from an area of high water concentration to an area of lower water concentration

2.

Driving force:

3.

Types of solutions a.

Hypertonic

b.

Hypotonic

c.

Isotonic

4.

“Water follows solutes”

5.

Response of blood cells to osmotic stress a.

Crenation

b.

Hemolysis

52 D.

III.

IV.

Filtration 1.

Def.: Movement of particles or molecules through a porous barrier

2.

Filtrate

3.

Driving force:

LAB AND SAFETY PROCEDURES A.

Use caution with all reagents. Do not allow the silver nitrate solution to splash; it stains the skin and is an irritant. Do not allow any reagent to contact your eyes; open bottles with rubber stoppers, such as the iodine, methyl orange dye, Evans blue dye, and the india ink, carefully. All of these can stain skin and clothing.

B.

Work in groups of 3-4 students for all procedures and for discussion of results.

C.

Take the reagents that you need from the large containers on the side benches by pouring the quantity you need into a smaller container and bringing it back to your work area. Label all experimental containers accurately. Do not return leftover reagents to the large containers; instead discard them down the sink.

DIFFUSION A.

Materials Beakers, hot water, cold water, india ink, prepared agar plates, cork borers, methyl orange stain, Evans blue stain, centimeter ruler.

B.

Procedures 1.

Effect of temperature on the rate of diffusion a.

Fill one 250-ml beaker half full of ice-cold tap water. Fill a second 250-ml beaker equally full of hot tap water. Allow each to settle for a minute or two.

53

2.

b.

Drop one drop of india ink into the center of each beaker, from the same height (about the level of the rim), as close together in time as is possible.

c.

Observe the diffusion of the ink. In which beaker did the diffusion occur faster? (hot / cold)

d.

Repeat if necessary. Explain your results in terms of the driving force of diffusion.

Effect of size of the molecules on rate of diffusion a.

Take a prepared plate of agar, a semi-solid substrate which is used for microbiological cultures.

b.

Using a cork borer, carefully remove two small disks of agar, leaving two round “wells” a few centimeters from each other and from the sides of the plate.

c.

Carefully fill one well with a drop of methyl orange stain. The molecules of methyl orange are known to have a molecular weight of 327, indicating that they are relatively small molecules.

d.

In the same way, carefully fill the second well with a drop of Evans blue stain. The molecules of Evans blue have a molecular weight of 961, indicating that they are relatively large, about three times more massive than the methyl orange.

e.

In 35- 45 minutes measure the diameter of the stained area around each well. In other words, measure from one edge of the stain across the well to the other side. Record below in mm. __________ diameter of methyl orange stain

f.

__________ diameter of Evans blue stain

Based on your observations, make a statement about the relationship between molecular size and rate of diffusion.

54 3.

V.

Does water diffuse? a.

Fill a 250-ml beaker with cold water. Let the water settle in it for a minute. Then, drop one drop of india ink into the beaker. Consider the ink. Where is the ink most concentrated as you begin? Where is the ink least concentrated? Does the ink move from high concentration of ink to lower concentration of ink? What will happen if the beaker is left untouched for a couple of hours? Why? What is the driving force for the movement of the india ink?

b.

Fill a second 250-ml beaker with cold water. Let the water settle in it for a minute. Again, drop one drop of india ink into the beaker. This time, consider the water. Where is the water most concentrated as you begin? Where is the water least concentrated as you begin? Does water move from high concentration of water to lower concentration of water? What is the driving force for the movement of the water? Does the water behave any differently than does the ink?

OSMOSIS A.

Materials Distilled water, 250 ml beakers, grease pencils,10% sucrose solution, 20% sucrose solution, dialysis tubing, scissors, string, wash bottle of 10% sucrose solution, electronic scale

B.

Procedure 1.

Fill and carefully label three beakers as follows: Beaker 1: distilled water Beaker 2: solution of 10% sucrose Beaker 3: solution of 20% sucrose

2.

Prepare three dialysis sacs by tying the lower end of a wet, 12 cm strip of dialysis tubing with a short piece of string. Using the wash bottle of 10% sucrose solution, fill the sacs you have made about 1/3 full.

55 3.

Once each sac is 1/3 filled, press all air out from the top and tie the top closed with string. Cut the string, leaving one end about 10 cm long. Rinse the sac and string by dipping them into a beaker of distilled water.

4.

Gently pat each sac with a paper towel to remove excess water. Weigh each sac and record the weight on the table below to the nearest 0.1 gm.

5.

Immerse one sac into each of the beakers prepared in step 1. Hang the string over the lip of the beaker, making sure the entire sac is immersed.

6.

Allow the beakers with sacs to remain untouched for 30 minutes.

7.

At the end of 30 minutes, use the string to remove each sac from the beaker. Write down any observations in size, color, or temperature that you note on the table below.

8.

Gently pat each sac with a paper towel, as before, to remove excess water. Reweigh each sac and record the weight on the table below.

9.

Calculate percent change in weight of each sac by subtracting the greater weight of each from its lower weight, and expressing that figure as a percentage of its original weight. A negative number indicates weight loss. For example, if the original weight was 10 g, and the final weight was 8 g, the difference is 2 g. The percent change, 2/10, or 20%, is negative, as the weight went down; thus, -20%. Or, if the original weight was 10 g, and the final weight was 14 g, the difference is 4 g. The percent change, 4/10, or 40% is positive; there was a 40% increase in the weight.

Sac 1 (Beaker 1) distilled water

Sac 2 (Beaker 2) 10% sucrose

Sac 3 ( Beaker 3) 20% sucrose

Observations

___________

______________

______________

Weight (start)

___________

______________

______________

Weight (end)

___________

______________

______________

Difference(+/-)

___________

______________

______________

% change (+/-)

___________

______________

______________

56 C.

Discussion 1.

Let each of the three sacs represent a cell; let the beakers represent various concentrations of solutions. a.

Which solution is hypertonic to the “cell”?

b.

Which solution is hypotonic to the “cell”?

c.

Which solution is isotonic to the “cell”?

d.

Which sac best illustrated crenation?

e.

Which sac best illustrated hemolysis?

2.

Why must there be a semipermeable membrane for osmosis to occur?

3.

It is known that a solution of 0.9% NaCl in water is isotonic to blood cells. a.

In a solution of 2.5% NaCl, which is similar to the concentration of sea water, will cells gain or lose water? Is this crenation or hemolysis?

b.

In a solution of 0.5% NaCl, will cells gain or lose water? Is this crenation or hemolysis?

c.

Since a 0.9% solution of NaCl is isotonic to blood cells, does that mean that blood cells contain 0.9% NaCl? (Y/N) Explain.

57 VI.

FILTRATION A.

Materials Ring stand, funnel, 250-ml beakers, test tubes, filter paper, distilled water bottle, silver nitrate, iodine solution; prepared “filtration mixture” of charcoal, a green dye, sodium chloride, and starch; stopwatch

B.

Procedure 1.

Set up the ring stand and set the funnel in it, leaving plenty of room below it to set a 250 ml beaker. Fold a filter paper into quarters and fix in the funnel with a little distilled water.

2.

From the side lab bench, take about 100 ml of the “filtration mixture,” containing water, charcoal, starch, dye, and sodium chloride. (Before you pour it, agitate the container to mix the contents.)

3.

Pour the filtration mixture carefully into the filter paper, being careful that none goes over the top of the paper. Once the rate of flow of the filtrate slows enough to count the drops, use the stopwatch and count the number of drops for 30 seconds. Pause for 30 seconds, and then count the number of drops for another 30 seconds. Pause for 30 more seconds, and then count the drops for 30 more seconds. Record your results below:

_______ drops per first 30 sec

_______ drops per second 30 sec

_______ drops per third 30 sec

Explain the difference in the rate of filtrate formation based on the driving force of filtration.

58 4.

5.

Collect the filtrate from the beaker. Evaluate its contents: a.

Use the color of the charcoal to determine if it filtered. (Y/N) Explain why or why not based on its ability to dissolve in water.

b.

Use the color of the dye to determine if filtered. (Y/N) Explain.

c.

Test for the presence of sodium chloride in the filtrate by using the silver nitrate test at the side lab bench. Carefully mix about 2 ml of the filtrate with several drops of the silver nitrate solution. A whitish precipitate is a positive test for sodium chloride. (Y/N) Explain.

d.

Test for the presence of starch by carefully adding a drop or two of iodine to the filtrate remaining in the beaker. A blue or purple color is a positive test for starch. (Y/N) Explain.

Carefully discard and rinse all used-up material down the sink. Do not return excess reagents to the large containers from which they came.

59 C.

Discussion 1.

What would be the effect on the rate of filtration if a pumping force (pressure) could be applied to the top of the funnel? Why?

2.

What would be the effect on the rate of filtration if the pores of the filter were larger? Smaller? Explain.