Diffusion, osmosis, transport mechanisms 43

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1 Diffusion, osmosis, transport mechanisms 43 DIFFUSION, OSMOSIS AND TRANSPORT MECHANISMS The cell membrane is a biological membrane that separates the interior of all cells from the outside environment and thus controls the movement of substances in and out of cells. It consists of the phospholipid bilayer with embedded proteins and is selectively-permeable to ions and organic molecules. All lipid-soluble substances can move freely through the membrane whereas water-soluble molecules can be transported passively or actively by the embedded protein. Thus the cell membrane plays an important role in determining the internal environment (homeostasis) of the cell. Water molecules can move through a selectively permeable membrane (a membrane that allows for diffusion of solutes) from a region of higher water potential (high concentration) to a region of lower water potential (low concentration) until an equilibrium is reached. DIFFUSION IN AGAR CELLS The aim of the laboratory practice is to determine how the rate of diffusion varies with the ratio of surface area to volume. Diffusion is the spontaneous movement of molecules or other particles in solution, owing to their random thermal motion, to reach a uniform concentration throughout the solvent, a process requiring no addition of energy to the system. The Brownian motion of large molecules is observable under a microscope; small-molecule diffusion can only be probed in carefully controlled experimental conditions. Under normal conditions, molecular diffusion is relevant only on length scales between nanometer and millimeter. On larger length scales, transport in liquids and gases is normally due to another transport phenomenon, convection. PRINCIPLE The phenolphthalein in the agar cubes reacts with the NaOH, changing the color of the cube to pink. After the cubes are exposed to NaOH, the color change indicates how far the NaOH diffused. This helps to determine the relationship between diffusion rate and the surface area and volume of the cubes. MATERIALS cubes of 3% agar-phenolphthalein (1 cm, 2 cm, and 3 cm on a side) 4% NaOH solution ruler blade paper towel beaker white tile Phenolphthalein can cause cancer. Do not swallow the cubes!

2 44 Physiology laboratory exercises PROCEDURE 1. Use three cubes (one of each: 1 cm, 2 cm, 3 cm). 2. Pour 4% NaOH solution into the beaker (at least 4 cm height). Place the cube with the 1 cm side into this solution. After exactly three minutes remove it and place it on the white tile. Using the blade cut the cube in three pieces using two parallel cuts. Use only the middle piece of the cube and measure the distance of the inner, uncolored part (b on the following image). Do the measurements quickly, as diffusion does not stop when you remove the cube. Using the table below calculate the surface and volume of the cubes. Repeat this procedure with a cube that has 2 cm sides and with one cube that has 3 cm sides. 3. Put 5 cubes with 2 cm sides in the NaOH solution. After one minute remove 1 cube, place it on the white tile and cut it into three pieces (with two parallel cuts). Using the middle piece measure the distance of the outer, colored part of the cube on one side (c on the image above). Note the value in the second table below. Repeat the procedure with the remaining cubes at exactly 2, 4, 8 and 16 minutes respectively (measured from the initial moment of introduction of the cubes in the solution). RESULTS Record the data you gathered during part 2 of the procedure in the following table. Cube size 1 cm / side Surface area Volume Surface area / volume ratio Size of uncolored portion of cube (side lenght) Volume of uncolored portion Volume of colored portion Ratio of the volume of colored portion 2 cm / side 3 cm / side

3 Diffusion, osmosis, transport mechanisms 45 The rate of diffusion is the penetration depth of NaOH molecules divided by the duration of the experiment. Calculate the rate of diffusion for each of the cubes. Cube #1: Cube #2: Cube #3: Record the data you gathered during part 3 of the procedure in the following table. Duration 1 minute 2 minute 4 minute 8 minute 16 minute Broadness of the colored part (c) CONCLUSIONS How does the surface area to volume ratio of the cube depend on the length of the side of a cube? How does the rate of diffusion depend on the surface of the cube? Why is the size of the cells limited? Why many cell organelles have folded membranes? DIFFUSION ACROSS A SELECTIVELY PERMEABLE MEMBRANE The aim of the laboratory practice it to observe the diffusion of a substance across a selectively permeable membrane. A semi-permeable (or selectively permeable) membrane allows the solvent and small solute molecules to diffuse across the membrane, while it is impermeable to larger molecules. PRINCIPLE Iodine solution (iodine-ki reagent) in the presence of starch (amylose) changes to a blueblack color. If starch amylose is not present, then the color will stay orange or yellow. MATERIALS plastic bag spoon starch

4 46 Physiology laboratory exercises iodine or Lugol s solution beaker distilled water pipette Iodine is toxic and will make a permanent stain on your clothes! PROCEDURE Fill a plastic bag with a teaspoon of starch and around 25 ml of water. Tie the bag. Fill a beaker halfway with water and add ten s of iodine. Place the bag in the cup so that the starch mixture is submerged in the iodine water mixture. Wait 60 minutes and record your observations. RESULTS Solution in beaker Solution in bag Starting color Color after 60 minutes CONCLUSIONS Based on your observations, which substance moved, the iodine or the starch? The plastic baggie was permeable to which substance? Is the plastic baggie selectively permeable? Why is iodine called an indicator? DETERMINATION OF THE OSMOTIC FRAGILITY OF THE RED BLOOD CELLS Osmotic fragility (or osmotic resistance) of red blood cells represents their sensitivity to changes in osmotic pressure. Osmosis is the diffusion of a solvent (water) through a semipermeable membrane (permeable to the solvent, but not the solute) separating two solutions of different concentrations. The osmotic pressure is defined to be the pressure required to maintain equilibrium, with no net movement of solvent. Osmolarity is the measure of solute concentration, defined as the number of osmoles (Osm) of solute per liter (L) of solution (osmol/l or Osm). Ionic compounds, such as salts, can dissociate in solution into their

5 Diffusion, osmosis, transport mechanisms 47 constituent ions. For example, sodium chloride () dissociates into Na + and Cl - ions. Thus, for every 1 mole of in solution, there are 2 osmoles of solute particles (i.e., a 1 M solution is a 2 Osm solution). Both sodium and chloride ions affect the osmotic pressure of the solution. Nonionic compounds do not dissociate and form only 1 osmole of solute per 1 mole of solute. For example, a 1 M solution of glucose is 1 Osm. Osmolarity and tonicity are related, but different, concepts. The terms are different because osmolarity takes into account the total concentration of penetrating solutes and non-penetrating solutes; whereas tonicity takes into account the total concentration of only non-penetrating solutes. If two solutions have the same osmolarity, they are said to be isosmotic to each other. If the two solutions are separated by a semi-permeable membrane, water will move between the two solutions, but there will be no net change in the amount of water in either solution. If two solutions differ in osmolarity, the more concentrated is hyperosmotic to the other. The solution that is less concentrated is hyposmotic to the one with more solute. These terms can only be used to compare solutions. If the two solutions are separated by a semi-permeable membrane, a net water flow will occur towards the hyperosmotic solution. In red blood cells suspended in an isosmotic solution (300 mosm, e. g. 0.9% solution) no visible modifications occur. Exposed to a hyposmotic solution, red cells take in increasing quantities of water, swell until the capacity of the cell membrane is exceeded, and burst releasing hemoglobin. Exposed to a hyperosmotic solution, red cells give up intracellular fluid and shrink. Osmotic fragility tests are based on the measure of red blood cell lysis as a function of osmotic stress (hyposmotic solution). PRINCIPLE The osmotic resistance of erythrocytes is determined by exposing erythrocytes to different concentrations of salt solutions. The hemolysis is observed after centrifugation as a light red colored supernatant. The osmotic resistance of the population of red cells is assumed to follow a normal probability distribution. MATERIALS 12 test tubes, 0.5% salt solution (0.5 g in 100 ml water, i.e. 5 g in 1 l water), distilled water, anticoagulant-treated blood. PROCEDURE Place the test tubes in a holder and mark them from 25 to 14. Put as many s of 0.5% salt solution in the test tubes as indicated by the marks. Complete with distilled water to 25 s. Mix the content of every tube (shake the tubes). Thus a series of hyposmotic concentrations of salt solution is obtained. Put one of packed red cells in every test tube and mix. Centrifuge the samples in order to speed up the sedimentation process.

6 48 Physiology laboratory exercises To avoid risk of infection, wash your hands with soap and water before and after doing any blood tests! When manipulating blood samples from another person, use disposable rubber gloves! Observe the signs of hemolysis! Observe the color of the supernatant and of the sediment! The hemoglobin released from the disintegrated red blood cells colors the supernatant in light red. Note the test tube with the less hyposmotic solution showing light red supernatant! If all of the red cells have been disintegrated, the sediment contains only the membranes and stroma of the red blood cells! Note the test tube with the less hyposmotic solution showing a small quantity of yellow sediment! RESULTS Calculate the concentration of the salt solution in the test tubes! m c V m c V c V V c No 25 V total total ; V 0.5 No c No 25 (%) Determine the saline concentration at which lysis begins! This represents the minimum osmotic resistance. Determine the saline concentration at which lysis is complete! This represents the maximum osmotic resistance. DATA INTERPRETATION Normal values: minimum osmotic resistance: 0.44% (4.4 g/l), maximum osmotic resistance: % (3-3.2 g/l) Higher values: increased osmotic fragility / decreased osmotic resistance: decreased surface area to volume ratio of erythrocytes (spherocytosis, elliptocytosis), altered red blood cell membrane (aging red blood cells due to decreased turnover, autoimmune hemolytic anemia). Lower values: decreased osmotic fragility / increased osmotic resistance: increased surface area to volume ratio of the red cells (thalassemia, iron deficiency).

Lab 4: Osmosis and Diffusion

Page 4.1 Lab 4: Osmosis and Diffusion Cells need to obtain water and other particles from the fluids that surround them. Water and other particles also move out of cells. Osmosis (for water) and diffusion