BI 101-709 Objectives: Lab Assignment #3: Diffusion and Osmosis Spring 2024 • To familiarize students with the processes of diffusion and osmosis • To investigate how thermal energy affects diffusion rate • • • • To investigate the influence of cell size (surface area to volume ratio) on the efficiency of diffusion into a cell To introduce the concept of water potential To describe isotonic, hypotonic, and hypertonic solutions, and explain how they relate to the movement of water into and out of cells and the consequences thereof To compare the effects of osmosis on animal cells with that on plant cells Introduction: All living organisms are composed of cells and all cells, whether they are prokaryotic or eukaryotic, have a plasma membrane that defines their boundary and controls what substances can be moved into and out of the cell. To stay alive, cells must be able to exchange substances with the environment, bringing nutrients in, and moving waste out. The direction of movement of these substances (either into or out of the cell) is very frequently determined by a process known as diffusion. Therefore, it is important for us to understand diffusion if we want to understand how cells maintain their inner equilibrium. Diffusion is simply the movement of atoms or molecules from an area of higher concentration (of them) to an area of lower concentration (of them). When there is a difference in concentration like that, we say there is a concentration gradient present. The gradient represents potential energy. Potential energy is the energy something has due to its structure (in the case of molecules) or location in space (such as a ball at the top of a hill). The movement of a substance from a higher to a lower concentration is like a ball rolling downhill: the steeper the hill, the faster the ball will roll; the steeper (greater) the gradient (concentration difference), the faster the diffusion will occur. This movement continues until equilibrium is achieved and the molecules are distributed equally. At that point, it is important to remember that molecules are still moving around, but there is no net movement in any one direction. Atoms and molecules are always in motion. Even in a solid state like ice they are vibrating at least. Diffusion can be easily demonstrated by placing a drop of food coloring into a beaker of water: Fig. 1. The process of diffusion. This movement is a consequence of the molecular motion of the atoms or molecules: it is a physical property of matter, regardless of whether that matter is 'alive' or not, and reflects the amount of thermal energy present in that matter. Thermal energy is what we call the kinetic energy associated with the random movement of atoms or molecules. Kinetic energy is the energy of motion, in other words. The faster the atoms or molecules are moving, the greater the thermal energy. Thermal energy in transfer from one object to another is called heat. Have you ever put your warm hand on a cold surface? In which direction does the 'heat' move? From your hand to the colder surface, right? Thermal energy moves from the area of greater thermal energy to the area of lesser thermal energy. Sounds like diffusion in a way: going from higher to lower. The same can be seen in our weather: wind (the movement of air molecules) blows from areas of higher pressure to areas of lower pressure. The steeper the pressure gradient, the stronger the wind. 1 A difference in the concentration of a molecule outside of a cell compared to the inside of a cell (a concentration gradient) can be the driving force behind movement of that molecule into or out of the cell (depending on which side has the higher concentration). If the molecule is small and nonpolar, it may diffuse directly through the cell membrane by itself (it has to be nonpolar so as to be compatible with the nonpolar, hydrophobic interior of the phospholipid bilayer that is the membrane). Larger and/or polar molecules that are present in a gradient situation will still move down their gradient into or out of the cell, but will require the assistance of membrane proteins that form a channel for them. That is called "facilitated diffusion" and is still a passive process, since the driving force is the concentration gradient. No energy is required from the cell to move the molecules down their own gradient. Refer to chapter 3, sections 4, 5, and 6, of our textbook for extra help, if needed. Part #1: Rate of Diffusion in a Liquid as a Function of Thermal Energy Would you predict that molecules with more thermal energy (i.e., a higher temperature) would diffuse faster, or more slowly, than molecules with less thermal energy (i.e., a colder temperature)? State your prediction here (in the form of a full sentence, with a period at the end of your sentence): The following exercise will allow you to test your prediction. Materials required: (Note: If you do not have access to these materials, use the video in the lab folder.) Two clear glass or plastic containers, such as drinking glasses or jars, of the same size Food coloring Hot and cold tap water 1. Watch the video "Diffusion of Food Dye in Hot and Cold Water" in the lab folder to get an idea of what you are going to do. 2. Label your containers "A" and "B." 3. 4. 5. 6. Fill container A with very hot water (about three fourths full), and container B to the same level with very cold water. The greater the temperature difference between them, the more dramatic the difference in your results will be. Place the containers on a counter or table where they will not be disturbed and allow them to sit for about five minutes so that the water no longer appears to be moving inside them. It is important that they are not disturbed during the experiment. We want the only movement of molecules to be from their own thermal energy, and not from outside influences such as shaking or moving of the containers. After those five minutes, when you think the water has stabilized, place a single drop of food coloring (your choice of color, but it must be the same color in each container) onto the surface of the water in each container. Do not move the containers or stir the water in any way. Observe the food coloring as it diffuses in the water in each container. Describe here what you see: Analysis: 1. Do your observations support the prediction you made? 2. In this experiment, what is the independent variable? 3. What is the dependent variable in this experiment? 2 4. Why was it important to have the containers be the same size, to have the amount of water in them be the same, and to have the same color dye placed in each container? 5. After you have added the drop of dye, which molecules (dye, water, or both) are moving at the beginning of the experiment? (Think carefully... the motion may not be apparent to the eye.) 6. Which molecules (dye, water, or both) are moving at the end of the experiment? Part #2: Modeling Diffusion in a Cell: Surface Area to Volume Ratio As you have learned, cells are very small. Have you thought about why cells are so small? Why can't they be bigger? One of the most important factors that limit how big a cell can be is the rate of diffusion. Cells must be small enough so that there is sufficient diffusion of ions and molecules into and out of them, as well as throughout their internal volume of cytoplasm. Those ions and molecules are required so the call can maintain homeostasis, the dynamic balance of biochemistry that supports life. Nutrient molecules must be brought in, waste molecules removed, and salt concentrations and pH controlled. Answer this question: in Part #1 of this lab exercise, you investigated the effect of temperature on the rate of diffusion and observed that higher temperature results in faster diffusion. Why can't cells simply increase their temperature to increase diffusion, and therefore be able to be bigger in size? [Do not look this up on the internet. Just think about it and write an answer in your own words.] In this part of the lab exercise, you will complete an exercise on Pivot Interactives that demonstrates the importance of the surface-area-to-volume ratio as a determinant of diffusion efficiency, and, therefore, allowable cell size. In general, cell size is limited because cell volume increases much more quickly than does cell surface area, as a cell gets bigger. To illustrate this fact, refer to Figure 2 below. If we use a cube as a model for a cell, the surface area increases as the square (cm²) of the length of the cube (surface area = area of one side times the number of sides), but the volume increases as the cube (cm³) of that dimension (length x width x depth). As a cell becomes larger, it becomes more and more difficult for the cell to acquire sufficient materials to support the processes inside the cell, because the amount of surface area (across which materials must be transported) relative to the volume of the cell declines. In other words, the surface-area-to-volume ratio decreases. 3 1 cm 2 cm 3 cm Cube Side Length Surface Area Volume Surface-area- to-volume ratio 1 cm 6 cm² 1 cm³ 6 cm 1 2 cm 24 cm² 8 cm³ 3 cm 1 3 ст 54 cm² 27 cm³ 2 cm-1 Volume increases at a higher rate than surface area units of 10000 measure 8000 a² V3 6000 4000 2000 total surface area volume 0 1 3 5 7 9 11 13 15 17 19 length of side Fig. 2. Comparison of the surface area to volume ratios of model cells of increasing size. To help you understand what you will be observing in the videos in the Pivot Interactives activity, look at Figure 3. Vinegar Agar cubes Fig. 3. Agar cubes of various sizes soaking in vinegar. Agar is a gel-like substance (like gelatin but made of carbohydrate instead of protein) derived from algae that allows diffusion in and out of it. The cubes in Figure 3, and in the Pivot exercise (the link to the exercise is in the lab folder) are made of an agar that also contains an indicator molecule that changes color when exposed to a change in pH. Vinegar is an acid. As the vinegar diffuses from the surrounding liquid into the agar cubes, the color indicator molecule will react with the acid, and we will be able to see the color of the cubes change, from the outside to their center, as the vinegar diffuses in. Now go to Pivot Interactives (using the link in the lab folder) and complete the activity there. When you are done with the Pivot activity, come back here and complete Part #3. 4 Part #3: Observing the Effects of Osmosis on Plant Cells Introduction: Diffusion is one of the processes by which substances such as nutrients, water, oxygen, and cellular wastes are exchanged between living cells and their environment. One of the most important molecules that must get in and out of cells is water since it is the solvent of life. The direction of movement of the water (into or out of the cell) is determined by differences in the concentration of water on each side of the plasma membrane (diffusion, in other words). This also describes differences in water potential. Water potential is the potential energy that water has per unit volume relative to pure water. Water potential quantifies the tendency of water to move from one area to another due to osmosis, gravity, mechanical pressure, etc. There is a mathematical equation that is used to calculate water potential, but for our purposes, we can just think of it as the potential that water has to do something, like diffuse from one area to another. Pure water has a higher water potential than water containing solutes. Osmosis is the term for the diffusion of water across a selectively permeable membrane (such as the plasma membrane). Like all molecules, water follows its concentration gradient and moves from an area of higher water concentration (higher water potential) to an area of lower water concentration (lower water potential). The movement of water into and out of a cell has very important consequences for the cell's structure and ability to function properly. Due to the phospholipids that make up the basic structure of a cell membrane, those membranes are selectively permeable: in other words, they allow some types of molecules to diffuse directly through (if those molecules are small and nonpolar), but not others. Tonicity is the term used to describe the relative concentration of solute, and therefore also of solvent (water), outside the cell compared to inside the cell. Fig.3.22 from our textbook (below) illustrates what happens to red blood cells when they are placed in solutions of different tonicities. Hypertonic solution Isotonic solution Hypotonic solution H₂O H₂O HO H₂O Figure 3.22 Osmotic pressure changes the shape of red blood cells in hypertonic, isotonic, and hypotonic solutions. (credit: modification of work by Mariana Ruiz Villarreal) A hypertonic solution ("hyper" means "over") has a higher concentration of solute (therefore, lower concentration of water) than the cell. When cells are placed in a hypertonic solution, water moves out of the cell into the surrounding hypertonic solution (where there is less water and less water potential), and the cell therefore shrinks in size. In terms of water potential, we would say that the water potential is higher inside the cell than outside, so the net movement of water is from inside the cell to outside the cell. 5