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Diffusion, Osmosis, and Tonicity

Human Physiology BIOL 2134L

Lab was modified from Exercise 2.6 in A Laboratory Guide to Human Physiology by Stuart Ira Fox, and Exercise 4 in Laboratory Manual for Understanding Human Anatomy and Physiology: Cat version by Stalheim-Smith, A., Gaines, R., and Robinson, S.; West Publishing Company 1993; pgs 29 ­ 34.

Equipment for Activity 5 Equipment for Activity 1

­ ­ ­ ­ Dialysis Tubing, 8 ­ 10 cm long String/yarn/dialysis tubing clamps 5, 250 mL Beakers Syrup/corn syrup/molasses/honey ­ in 5%, 25%, and 50% solutions (with NaCl [0.5%, 2.5%, and 5.0% respectively] added) Sodium chloride (NaCl) solution ­ in 5%, 10%, and 25% solutions Scale 10 10-mL test tubes Test tube racks Silver nitrate (AgNO3) Potassium dichromate (K2Cr2O7) Disposable pipettes ­ ­ ­ ­ ­ ­ Animal blood Sodium chloride solutions in dropper bottles in these concentrations: 0.20 g/100 mL; 0.45 g/100mL;0.85 g/100 mL; 3.5 g/100 mL;and 10 g/100 mL Microscope slides Microscope cover slips Microscopes

­ ­ ­ ­ ­ ­ ­

Equipment for Activity 6

­ ­ ­ ­ ­ ­ ­ ­ Scale 4 ­ 150 mL beakers 1 ­ 250 mL beaker 1 ­ 100 mL graduated cylinder 14 oz beads 20 oz shells 2 weighing boats tap water

Equipment for Activity 2

­ ­ ­ ­ 1% carmine red suspension (in solution) Microscope slides Microscope cover slips Microscope

Equipment for Activity 3

­ ­ ­ ­ ­ Methylene blue (powder) Malachite green (crystals) 2 Fine-tip forceps 2 50 mL beakers with room temperature water 2 50 mL beakers with chilled water

Osmosis is the net diffusion of water (solvent) through a membrane that separates two solutions. Osmosis occurs passively when the two solutions have different total concentrations of molecules (solutes) to which the membrane is relatively impermeable. If there is no osmosis when a membrane separates two solutions, those solutions are said to be isotonic to each other.

OBJECTIVES Equipment for Activity 4

­ ­ ­ ­ ­ Methylene blue (powder) Malachite green (powder) Fine-tip forceps Petri dish - room temperature Petri dish - chilled 1. Distinguish between the terms solute, solvent, and solution. 2. Define the terms passive transport, diffusion, and active transport. 3. Define the terms osmosis, osmotic pressure, and osmolality.

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4. Define the terms isotonic, hypotonic, and hypertonic. 5. Calculate the osmolality of solutions when the concentration of solute (in g/L) and the molecular weight of a solute are known. 6. Describe how red blood cells (RBCs) are affected when they are placed in isotonic, hypotonic, and hypertonic solutions. If you were to drop a pinch of sugar (the solute) into a beaker of water (the solvent), the resulting solution would, after a time, have a uniform sweetness. The uniform sweetness would result from the constant state of motion of all the solute and solvent molecules in the solution, producing a net movement of solute molecules from regions of higher concentration to regions of lower concentration. This net movement of solute molecules is called diffusion. The rate of diffusion is proportional to the concentration differences that exist in the solution. The diffusion rate will steadily decrease as the solute becomes evenly distributed in the solvent, and diffusion will cease entirely when the solution becomes uniform. A molecule may move into or out of a cell by diffusion is (1) a difference in the concentration of that molecules (concentration gradient) exists between the intracellular and extracellular compartments, and (2) the plasma (cell) membrane will allow the passage of that molecule. The movement of a molecule across the plasma (cell) membrane by diffusion is called passive transport. The term passive is used because the cell need not expend energy in the process (no ATP is used). By contrast, cells often must move molecules across the plasma 9cell) membrane from lower to higher concentrations; that is, cells must "fight" diffusion in the attempt to maintain a concentration difference across the 2

membrane. However, to move molecules "up-hill" against their concentration gradients, the cells must expend energy; they must use ATP. This process is called active transport. The permeability of a membrane refers to the ease with which substances can pass through (permeate) it. A membrane that is completely permeable to all molecules is not a barrier to diffusion, whereas a membrane that is completely impermeable to all molecules essentially divides the solutions into two non-communicating compartments. Since a living cell must selectively interact with its environment, taking in raw products and excreting waste products, the cell is surrounded by a membrane that is semi- (or selectively) permeable. A semi-permeable membrane is completely permeable to some molecules, slightly permeable to others, and completely impermeable to still others. The plasma (cell) membrane is composed primarily of two semi-fluid phospholipid layers with proteins. Some proteins are partially submerged; others span the complete thickness of the membrane. In this way, the membrane is not continuous but behaves as if tiny protein channels were serving as waterways for diffusion, allowing passage of ions and smaller molecules while excluding the passage of molecules larger than the channels.

Osmosis

The diffusion of water is a special case which deserves attention because water is the major component of the intracellular and extracellular fluids of your body. The net movement of water across a selectively permeable membrane from a region of high water concentration to one of lower water concentration is osmosis. Water will cross plasma membranes into cells if the water concentration is higher in

the extracellular fluid than in the intracellular fluid.

A

H 2O

Water concentration is higher (more dilute = lighter blue) outside the cell. Water concentration is lower in the cell (less water = darker blue). Water will cross the plasma (cell) membranes out of the cells if the water concentration is higher in the intracellular fluid than in the extracellular fluid.

B

H 2O

Water concentration is lower outside the cell (less water = darker blue). Water concentration is higher (more dilute = lighter blue) inside the cell. The flow of water across a selectively permeable membrane can be stopped if sufficient pressure opposes this flow. The pressure required to prevent the flow of water across a membrane is called osmotic pressure. The concept of osmotic pressure can be seen in the figure below.

Osmosis Hydrostatic pressure

Figure taken from http://hyperphysics.phyastr.gsu.edu/hbase/kinetic/imgkin/osm2.gif

In this figure, a dilute sugar solution (less sugar and more water) is on the lefthand compartment of the membrane in part A and a concentrated sugar solution (more sugar than water) is on the right-hand compartment of the membrane. (· = water or the solvent, = solute or the sugar). The sugar molecules are too large to penetrate the membrane. In this figure, net water flow will occur from the dilute sugar solution to the concentrated sugar solution. What happens in the right-hand compartment as it receives more water? The increase in volume in this compartment creates a difference between the two compartments in the fluid (hydrostatic) pressure on the membrane. This hydrostatic pressure difference opposes the diffusion of water into the right-hand compartment. The hydrostatic pressure that completely stops osmosis is equal to the osmotic pressure of the solution in the right-hand compartment. The important thing to remember about this concept is that as the concentration of 3

solute (in this case sugar) in the right-hand compartment rises, the osmotic pressure of that solution increases (you need a larger volume of water in the right-hand side of the compartment to keep additional water from entering the right-hand compartment). Related to the concept of osmotic pressure is the concept of tonicity. The tonicity of a solution describes the effect which the solution has on a normal cell. If cells do not undergo any change in volume when added to a particular solution, that

solution is said to be isotonic. If cells undergo crenation or swelling or bursting or lyse (due to increases in cell volume) when added to a solution, the solution is hypotonic. If cells undergo shrinking or shriveling (due to decreases in cell volume) when added to a solution, the solution is hypertonic.

Figure taken from http://fig.cox.miami.edu/~cmallery/150/memb/sf4x12.jpg

Because the function of cells in the body is disturbed if they swell or shrink, the tonicity of the extracellular fluid is regulated at all times by controlling its relative amounts of solutes and water. Osmosis, osmotic pressure, and tonicity will be demonstrated by several activities in this lab exercise. In diffusion and osmosis, the movement of materials is powered by kinetic energy due to molecular motion ­ that is, by the heat of the surrounding environment. However, another agent, hydrostatic pressure, can move substances across a 4

barrier. The process by which ions or molecules are forced across a barrier in response to hydrostatic pressure is filtration. Pouring a solution into a funnel lined with filter paper is an example of filtration. In this example, the pressure that drives filtration is supplied by gravity. In the body, the pressure that drives filtration is blood pressure, which is supplied by the contractions of the heart. The barrier across which filtration occurs is formed by the walls of the capillaries, which are the smallest blood vessels. In the kidneys, for

example, components of blood in the kidney capillaries are forced through the capillary walls and into the surrounding fluid by blood pressure. The capillary wall acts as a filter, in that it bars the crossing of some but not all substances. Any substance in the blood that is small enough to pass through small pores in the capillary wall is filtered out of the blood. Substances in the blood that are too large to pass through the capillary pores, for example, blood cells and large plasma proteins, remain in the blood. NOTE: Dialysis tubing is an artificial membrane with microscopic pores which are similar in size to those in the kidney. A. Before making and filling you dialysis tube, pour the sodium chloride solutions and distilled water into the larger beakers set out for you. Then, using a disposable B. Obtain five 8 ­ 10 cm lengths of dialysis tubing and tie one end tightly with string/yarn/dialysis tubing clamps.

Activity 1 ­ Osmosis and Diffusion across a selectively permeable membrane

Focus questions: · How does the concentration gradient for a specific molecule affect diffusion across a selectively permeable membrane? · How does molecular size affect diffusion across a selectively permeable membrane?

» You can make a tighter knot if you

first fold the end of the tubing into small "accordion pleats".

Figure taken from http://pirate.shu.edu/~rawncarr/osmolab/bags1.gif

C. Fill each tube as directed in the table below. Each Group may be assigned specific solutions instead of each group doing all six (6) solution combinations.

Bag Number

1 2 3 4 5 6

Solution in Dialysis Bag

Distilled water (dH2O) Distilled water (dH2O) Distilled water (dH2O) 5% Syrup 25% Syrup 50% Syrup

Solution or Dialysate in Beaker

5% Sodium Chloride (NaCl) Solution 10% Sodium Chloride (NaCl) Solution 25% Sodium Chloride (NaCl) Solution Distilled water (dH2O) Distilled water (dH2O) Distilled water (dH2O)

» Rubbing the collapsed tubing between your fingers under running tap water is an easy way

to open the tubing up. You can then dump the water out and fill the bag with the solutions indicated above in the table. D. Squeeze out as much air as possible in each bag and then tie or clamp the open end of the tube. Be sure to leave 0.5 ­ 1 cm of empty space in the bag. E. Rinse the outside of the bag with distilled water. Then thoroughly dry the bag with paper towels and weigh 5 the dried bag. Record the weight in the Results. F. Before placing the bag in the dialysate you need to calculate the grams of NaCl in the dialysate. Follow the table below for how to do this:

NOTE: Be very careful not to mix any of the stock solutions and/or eyedroppers during the following procedures. The silver nitrate (AgNO3) used in the following test can damage clothing and discolor skin. Use care when handling the dropper bottle of silver nitrate. You should wear exam gloves while analyzing your urine samples. a. See table below to determine how much dialysate and how much distilled water you need to add to each test tube.Use a disposable pipette for dialysate and distilled water (use a separate pipette for each sample). b. Add 1 drop of 20% potassium dichromate (K2Cr2O7) to the test tube containing the dialysate and swirl to mix. From now on the contents of this test tube will be called the dialysate mixture. c. To measure the chloride ion (Cl-) concentration of the dialysate mixture, you will determine the number of drops of a 2.9% silver nitrate (AgNO3) solution which must be added to change the color

of the dialysate mixture from yellow to brown. Add 1 drop of AgNO3 to the test tube, letting it drop freely into the dialysate mixture, and not along the side of the test tube. Swirl to mix. Continue adding drops of AgNO3, one drop at a time, and mixing after each addition until the color of the dialysate mixture stays brown after mixing. Record the number of drops of AgNO3: d. Since each drop of AgNO3 reacts with the equivalent of 1 gram/liter (g/l) of sodium chloride, the number of drops of AgNO3 indicates the salt concentration. For example, if it took 7 drops to change your sample from yellow to brown, the salt concentration was 7 g/l. Record the salt concentration of the dialysate mixture in the Table below. e. Since we diluted the dialysate for bags 1, 2, and 3 with distilled water, you need to multiply the salt concentration in step d by 2.

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# drops of AgNO3 Dialysate for bag # 1 ­ put 5 drops dialysate and 5 drops of distilled water in test tube Dialysate for bag # 2 ­ put 5 drops dialysate and 5 drops of distilled water in test tube Dialysate for bag # 3 ­ put 2 drops dialysate and 8 drops of distilled water in test tube Dialysate for bag # 4 ­ put 10 drops dialysate (distilled water) in test tube Dialysate for bag # 5 ­ put 10 drops dialysate (distilled water) in test tube Dialysate for bag # 6 ­ put 10 drops dialysate (distilled water) in test tube BEFORE dialysis bag is added AFTER dialysis is complete BEFORE dialysis bag is added AFTER dialysis is complete BEFORE dialysis bag is added AFTER dialysis is complete BEFORE dialysis bag is added AFTER dialysis is complete BEFORE dialysis bag is added AFTER dialysis is complete BEFORE dialysis bag is added AFTER dialysis is complete

Salt Concentration X2= X2= X2= X2= X4= X4=

G. Place each bag in the beaker with the indicated solution. The bag must be H. You will record the Results table below.

completely submerged in the solution in the beaker.

Bag Solution in Number Dialysis Bag

Initial Weight of Bag

Weight of Bag after 15 minutes

Weight of Bag after 30 minutes

Weight of Bag after 45 minutes

Weight of Bag after 60 minutes

1 2 3 4 5

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dH2O dH2O dH2O 5% Syrup 25% Syrup 50% Syrup

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Activity 2 ­ Brownian Movement

Focus question: · How can the random movement of molecules be visualized? NOTE: In this activity you will be using a suspension which contains water and carmine red dye. The dye particles do not dissolve in water. If left to set, the dye particles would eventually settle out. When mixed, the small particles are suspended throughout the water. A. With an eyedropper, place one drop of carmine red dye suspension on a slide and cover the drop with a coverslip. Place the slide on a microscope. B. Using the scanning lens, bring the dye particles into focus. Switch to the medium-length objective lens and bring the particles into focus. C. Observe the Brownian movement of the dye particles. The particles may be flowing across your field of view ­ this is NOT Brownian movement. Brownian movement is the "jiggling" of the particles as they flow. Brownian movement is also observable in dye particles that are not flowing. D. Describe your observations below.

B. Malachite Green is a green dye (molecular weight 929.0). C. Using the forceps for methylene blue, place a small pinch of methylene blue in a beaker with room temperature water. D. Using the forceps for malachite green, place a small pinch (the same size pinch as for the methylene blue in step C) of malachite green in a beaker with room temperature. E. Note the relative time it takes for each dye to diffuse in the room temperature water. Which dye is faster? Which dye is slower? Why?

F. Using the forceps for methylene blue, place a small pinch of methylene blue (the same size pinch as for the methylene blue in step C) in a beaker with chilled water. G. Using the forceps for light green, place a small pinch (the same size pinch as for the methylene blue in step C) of malachite green in a beaker with chilled water. H. Note the relative time it takes for each dye to diffuse in the chilled water. Which dye is faster?

Activity 3 ­ Diffusion in a Liquid

Focus questions: · How does molecular size affect the rate of diffusion? · How does temperature affect the rate of diffusion? A. Methylene blue is a deep blue dye (molecular weight 320). 8

Which dye is slower? Why?

Activity 4 ­ Diffusion in a gel

Focus question: · How does the rate of diffusion in a liquid compare to the rate in a gel? The consistency of intracellular fluid resembles that of gelatin more than it does water. Agar is a gelatinous substance. The rate of diffusion of substances through intracellular fluid more closely resembles the rate of diffusion through agar than through water. A. Using the forceps for methylene blue, place a small pinch of methylene blue on a room temperature agar plate. (Use one fourth (1/4th) the plate for the methylene blue and the other fourth (1/4th) for the malachite green.) B. Using the forceps for malachite green, place a small pinch (the same size pinch as for the methylene blue in step A) of malachite green on a room temperature agar plate. (Use other one fourth (1/4th) of the plate you used for the methylene blue.) C. Note the relative time it takes for each dye to diffuse in the room temperature agar. Which dye is faster? Which dye is slower? Why?

plate. (Use other one fourth (1/4th) of the plate you used for the methylene blue.) F. Note the relative time it takes for each dye to diffuse in the chilled water. Which dye is faster? Which dye is slower? Why?

Activity 5 ­ Tonicity and Red Blood Cells - Demonstration

Focus question: · How does the tonicity of a solution affect the shape, and ultimately the function, of red blood cells? A. Put 30 drops of each saline solution into different test tubes. B. Add 3 drops of animal blood to each test tube and gently mix the solution. C. Put 1 drop from each saline/blood solution on a microscope slide then add a cover slip. Look at the slide under the high dry power and observe the cells. D. Draw the red blood cells at each saline concentration. This activity may be set up as a demonstration.

D. Using the forceps for methylene blue, place a small pinch (the same size pinch as for the methylene blue in step A) of methylene blue on a chilled agar plate. (Use one fourth (1/4th) the plate for the methylene blue and the other fourth (1/4th) for the malachite green.) E. Using the forceps for malachite green, place a small pinch (the same size pinch as for the methylene blue in step A) of malachite green on a chilled agar 9

0.20 g/100 mL

0.45 g/100mL

D. Doing one 150 mL beaker at a time, pour the water of the beads or shells into the graduated cylinder. Record the volume of water for each here: _____ volume of water poured off the beads _____ volume of water poured off the shells Is the volume of water the same? 0.85 g/100 mL 3.5 g/100 mL If not, which beaker held more water? Which held less?

10 g/100 mL

Activity 6 ­ Molarity vs. Molality

Focus question: · How does the water concentration vary between different molecules for molarity and molality. · Molarity A. Place a weighing boat on the scale and weigh out 10 g of beads and pour the beads into a 150 mL beaker. (Add 10 g to the weight of the weighing boat to get to as close to 10 g of beads as possible.) B. Place a weighing boat on the scale and weigh out 10 g of shells and pour the shells into a second 150 mL beaker. (Add 10 g to the weight of the weighing boat to get to as close to 10 g of shells as possible.) C. Put water into the 250 mL beaker. Pour the water from the 250 mL beaker into the 150 mL beakers until each beaker reaches a total volume of 80 mL.

· Molality A. Place a weighing boat on the scale and weigh out 10 g of beads and pour the beads into a third 150 mL beaker. (Add 10 g to the weight of the weighing boat to get to as close to 10 g of beads as possible.) B. Place a weighing boat on the scale and weigh out 10 g of shells and pour the shells into a fourth 150 mL beaker. (Add 10 g to the weight of the weighing boat to get to as close to 10 g of shells as possible.) C. Using the graduated cylinder measure out 100 mL of water to pour in each 150 mL beaker. D. Record the final volume of fluid in each beaker. _____ volume of solution in the beaker with the beads _____ volume of solution in the beaker with the beads Is the volume of water the same? If not, which beaker had more solution? Which had less?

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Questions

1. Was there any change in the weight of the dialysis bags from the initial weight to the final weight? If so, compare the time for each bag (for instance time 0 after 15 min, after 30 min, after 45 min, after 60 min) for bag 1, then for bag 2, etc. Finally compare the final weights for all 6 bags to each other.

2. Was the final weight what you expected for each bag? Explain why or why not.

3. Make a separate line graph for each of the dialysis bags. On the graph plot time versus change in weight for each of the time periods. (Weight change is the original weight ­ the weight at each time interval.) From you graphs determine which time interval had the greatest weight change. Explain this observation.

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4. Make a line graph of each of the dialysis bags and their final weight change. (Weight change is the original weight ­ the final weight.) From your graph determine which solution had the greatest weight change. Explain this observation.

5. The solution placed in the beakers for dialysis bag #1, #2, and #3 contained a sodium chloride solution instead of distilled water. Did the salt concentration of the solution in the beaker change after the dialysis bag soaked in it for an hour? Did this result match what you expected to happen? Explain what was happening.

6. Suppose a salt and a glucose solution are separated by a membrane that is permeable to water but not to the solutes. The salt solution has a concentration of 1.95 g per 250 mL. The glucose solution has a concentration of 3.0 g per 250 mL. Will osmosis occur, and if so, in which direction? Explain your answer.

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7. When the body needs to conserve water, the kidneys excrete a hypertonic urine. What do the terms isotonic, hypotonic, and hypertonic mean? Draw beakers with red blood cells in them to demonstrate isotonic, hypotonic, and hypertonic.

8. Which component(s) of the syrup solution was/were osmotically active. Explain why this is true.

Syrup Ingredients

Corn syrup High fructose corn syrup Water Natural and artificial maple flavors Cellulose gum Caramel color Potassium sorbate (preservative) Citric acid Salt

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9. The dialysis membrane tubing we used for this experiment had pores that would permit molecules of a molecular weight of up to 13,000 to pass through it. Pores in the membrane permit the passage of water, most ions, and small molecules in solution. Particles of high molecular weight, such as polysaccharides, starch, and protein are too large to pass through the pores in the membrane. However, molecular weight is not the only determining factor that permits osmosis and diffusion. Other factors affecting molecular passage (transfer) or membrane permeability include molecular size and shape, pH of the solution used, tension on the membrane, and the length of time the test ran. For our experiment we placed the following molecules into the dialysis tubing. Molecule Water Sodium chloride (NaCl) Glucose Starch Albumen (protein) Molecular Weight 18 58.4 342 10,000 43,500

a. According to this, which of the molecules should have been able to pass through the dialysis tubing in our lab?

b. Did your results match the expected results? Why or why not?

10. The receptors for thirst are located in a part of the brain called the hypothalamus. These receptors, called osmoreceptors, are stimulated by an increase in blood osmolality. Imagine a man who has just landed on a desert island. Trace the course of events leading to his sensation of thirst. Can he satisfy his thirst by drinking seawater? Explain your answer.

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11. Before the invention of refrigerators, pioneers preserved meat by salting it. Explain how meat can be preserved by this procedure. (HINT: Think about what salting the meat would do to decomposer organisms, like bacteria and fungi.)

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