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Membrane Transport This online lab was adapted by David A. Lang, MS from the following: Biology Department, Glendale Community College. (2014). Membrane Transport. In BIO181: General Biology Laboratory Manual, 5/e (pp. 57–68). Glendale, AZ. Objectives · To become familiar with the process of diffusion and some of the factors that affect diffusion of substances across membranes · To learn more about the chemical nature of cell membranes Introduction Many activities of cells involve movement of materials across cell membranes. For instance, absorption, ingestion, respiration, photosynthesis, protein synthesis, and elimination of wastes all require membrane transport of materials into and out of cells. Molecules of matter tend to be in constant random motion. A particle moves in a given direction until it collides with another particle, at which time its direction of movement is changed. This random motion of particles is called Brownian Motion. As a result of Brownian Motion, particles of a substance tend to move from areas in which they have a greater activity to areas in which they have less activity. The activity of particles is influenced by concentration, temperature, and pressure. For biological systems, concentration of particles is frequently the most important influence. Particles move from an area of greater concentration to an area of lower concentration. This movement is called diffusion. When the concentration of particles becomes uniform, equilibrium has been achieved, and diffusion ceases. Random movement of particles, however, continues. Diffusion can occur in gases, in liquids, and in solids. It can occur across membranes. The purpose of this laboratory is to study some aspects of membrane transport involving diffusion. Part 1 – Movement of Materials Across Nonliving Membranes Many membranes are selectively or semipermeable, meaning some substances can readily diffuse across the membrane, while other substances cannot. The membrane is said to be permeable to those substances which can pass through the membrane, and impermeable to those which cannot. Because diffusion involves the random motion of particles, it is a passive process. The cell does not expend any energy to carry out diffusion. Some of these concepts can be demonstrated using an artificial membrane system. We will look at demonstration of a "model cell" as found at the link below. While not identical to the membranes of a living cell, this experimental setup allows for the control of the composition of the solutions both within and outside the “cell” and to thereby explore the process of diffusion and the concept of membrane permeability. View the demonstration: Cell Membrane Model Demonstration Using Dialysis Tubing https://www.youtube.com/watch?v=2CNGe3jMej0 Questions 1. Based on the results, what conclusions can you draw about the permeability of the artificial membrane? (Was it totally permeable, totally impermeable or selectively permeable?) 2. Which of the 3 solutes (iodine, fructose, and starch) were able to pass through the membrane (in or out)? 3. a) By what process did they cross the membrane? b) Why were the net movements of these substances in the directions they were? 4. a) ls energy required for this process? b) What is the experimental evidence? 5. a) Did the fructose molecules reach a state of equilibrium? b) What is the experimental evidence? 6. What is diffusion of water across a membrane called? 7. How do you think the results of this experiment would change if the experiment was done at a colder temperature? at a warmer temperature? (Assume that the membrane's permeability properties are unaffected by temperature). Explain. 8. a) Could it be that molecular size is important to the permeability of the artificial membrane? b) Explain giving evidence from the experiment. 9. On the basis of this experiment, what conclusions can you draw about the process of diffusion? 10. Compare and contrast the artificial membrane and the plasma membranes of cells. Part 2 – Osmosis Cell membranes are selectively permeable. They are permeable to water. This means that water can diffuse across cell membranes. Whether or not water will cross cell membranes depends upon the osmotic concentrations inside and outside the cell. The cytoplasm of cells consists of water in which are dispersed solutes and colloidal particles. The membrane is impermeable to many of these substances. The concentration of solutes and colloidal particles effects the movement of water into and out of the cell. Diffusion of water across membranes is called osmosis. The concentration of substances that effect osmosis is referred to as the solute or osmotic concentration of a cell. The concentration of water is inversely proportional to the concentration of solutes. Since water diffuses from an area of higher water concentration to an area of lower water concentration, it will diffuse from an area of lower solute concentration to an area of higher solute concentration. A cell that has a higher solute concentration than the surrounding medium is said to be hypertonic to its environment; a cell which has a lower solute concentration than the surrounding medium is said to be hypotonic to its environment; and a cell which is in equilibrium with the surrounding medium is isotonic to its environment. A. Estimation of Solute Concentration in Potato Clearly, then, the osmotic concentration is important in determining the osmotic activity of cells. lt is difficult to measure the osmotic concentration of cells or tissues directly, but indirect determination can be made. Tissue lengths of potato cores can be measured before and after submersion in solutions containing different concentrations of solutes (i.e., solutions of different concentrations). Cells or tissues that have a greater solute concentration than the solution in which they are submerged will gain water and, hence, increase in size. Such cells would be considered hypertonic compared to the surrounding solution, and the surrounding solution would be considered hypotonic compared to the cells. Cells that have a lower solute concentration than the surrounding solution will lose water and, hence, decrease in size. In this case, the surrounding solution would be considered hypertonic compared to the cells, and the cells would be considered hypotonic compared to the environment. Cells whose solute concentration is equal to that of the surrounding solution will be in equilibrium and will stay the same size. Such cells would be considered isotonic with the surrounding solution. View the demonstration: Osmosis in Potato Strips https://www.youtube.com/watch?v=jTDATlaBV-o Using the data collected during the demonstration, a data table was produced and is found below. Table 1. Effect of Salt Concentration of Surrounding Solution on Length of Potato Core Strips Salt Concentration (g/100mL) Initial Potato strip length (cm) Final Potato strip length (cm) Change in length (%) Average change in length (%) 0 6.0 6.1 1.67 3.89 6.0 6.2 3.33 6.0 6.4 6.67 1 6.0 6.1 1.67 1.11 6.0 6.0 0.00 6.0 6.1 1.67 2 6.0 5.9 -1.67 -1.67 6.0 5.9 -1.67 6.0 5.9 -1.67 3 6.0 5.9 -1.67 -3.33 6.0 5.8 -3.33 6.0 5.7 -5.00 4 6.0 5.7 -5.00 -4.44 6.0 5.7 -5.00 6.0 5.8 -3.33 5 6.0 5.7 -5.00 -4.44 6.0 5.7 -5.00 6.0 5.8 -3.33 Using the salt concentrations (left-hand column) and average changes length of potato strips (right-hand column), create a simple line graph (one line) using Excel (preferably) and paste the graph below. Independent variable is plotted on the x-axis and the dependent variable is plotted on the y-axis. Be sure to include your axis titles and graph title on your graph. Use this guidance video to help you: https://www.loom.com/share/5d2c25c3986545c3ae1563772e6b08b8 Although I prefer a graph competed and pasted from Excel, alternatively, you may hand-draw a graph using the graph paper on page 106 of your lab manual. Once completed, you could then take a picture of your drawing and then paste it below. Questions 1. If the potato cells were hypotonic to the surrounding solution, the surrounding solution itself would be what to the potato cells (hypertonic, hypotonic, or isotonic)? 2. Were the potato cells submerged in water with no salt hypertonic, hypotonic, or isotonic to the surrounding environment? 3. Were the potato cells submerged in 5 g/100mL salt solution hypertonic, hypotonic, or isotonic to the surrounding environment? 4. Look at your graph. At approximately what concentration of salt solution would the potato cells have likely been isotonic with the surrounding solution? [Tip: Estimate approximately at what concentration there would have been no change in the average length of potato strips.] B. Turgor and Plasmolysis You have observed that concentration differences between cells or between a cell and its environment can affect the movement of water across cell membranes. This can be observed using changes in volume or size as in the previous experiment or visually, looking at changes in appearance in the microscope, as you will do as you explore this experiment. The cells of plants are surrounded by a slightly elastic cellulose cell wall. If a plant cell is placed in a hypotonic solution (the cell has a greater solute concentration than the surrounding medium), water will diffuse into the cell. The contents of the cell begin to press against the cell wall and exert a pressure (called turgor pressure) against the wall causing the wall to stretch slightly. The cell wall exerts an equal but opposite pressure (wall pressure) against the cell contents. As more water diffuses into the cell, turgor pressure is increased; wall pressure is increased also. Water diffuses into the cell due to differences in solute concentration, but when the inward pressure exerted by the wall equals the tendency of water to diffuse in, diffusion stops even though a concentration equilibrium has not been reached. Thus in plant cells, concentration is not the only force which influences water movement. Pressure is also an important influence. Plant cells are normally turgid as turgor pressure causes the cell membrane to be firmly pressed against the cell wall, this provides a degree of rigidity for the cell. If the cell is placed in a hypertonic solution, the solutes do not readily cross the cell membrane, causing water to diffuse out of the cell. The loss of water reduces the volume of the protoplasm (everything from the cell membrane to inside the cell) causing the cell membrane to pull away from the cell wall. This phenomenon plasmolysis. View the demonstration: Plasmolysis in Elodea https://www.youtube.com/watch?v=c8nhJBw_So8 Procedure in the Demonstration Imagine you are preparing a wet mount of a young Elodea leaf. You use 3 drops of 5% NaCl to wet the slide with the young Elodea leaf and then place on the cover slip. Watch now what happens in the demonstration (video above). Questions 1. Did