Diffusion, Osmosis, and Membrane Transport

Diffusion, Osmosis, and Membrane Transport Introduction... 2 Diffusion and osmosis as related to cellular processes... 2 The hotter the medium, the faster the molecules diffuse... 2 TASK 1: TEMPERATURE AND THE RATE OF DIFFUSION... 2 The lighter the molecule, the faster it diffuses... 3 TASK 2: MOLECULAR WEIGHT AND THE RATE OF DIFFUSION... 3 The net rate of diffusion depends on the concentration gradient... 4 TASK 3: OSMOSIS, EFFECT OF CONCENTRATION ON THE RATE OF DIFFUSION... 4 Molecules diffuse across membranes depending on membrane permeability... 5 TASK 4: MOLECULAR SIZE AND MEMBRANE PERMEABILITY... 5 Plant Cells and Osmosis... 7 Tonicity... 7 TASK 5: POTATO CELLS AND OSMOSIS... 7 TASK 6: ELODEA CELLS AND OSMOSIS... 8 Summary... 9 General Biology 1 Instructor: Jose Bava, Ph.D

Introduction Diffusion and osmosis as related to cellular processes Cells constitute the units of life and in order to stay alive they need to fulfill their metabolic activities. Remember that metabolism is referred as the sum of the chemical reactions that take place within each cell of a living organism and that provide energy for vital processes and for synthesizing new organic material. Chemical reactions always involve molecules that are transformed into a different type of molecule, but before having the chemical reaction in a cell we need the molecules that participate in every reaction. How do all the reactants, molecules that participate in a chemical reaction, appear in the cells? Those that are not a product of a previous reaction need to enter the cell by moving across the cell membrane. Materials constantly move across the membrane, some examples are 1. Glucose and oxygen are needed inside the cell for aerobic cellular respiration to occur 2. Carbon dioxide leaves cells as a byproduct of aerobic cellular respiration 3. Plant cells need carbon dioxide to produce carbohydrates by means of photosynthesis. 4. Na+ (sodium) and K+ (potassium) ions moving into and out of nerve cells are mainly responsible for the generation of nerve impulses, an electrical occurrence that travels down a neuron. This electrical signal transmits commands from the brain to the motor nerves, and carries sensory messages from sensory nerves (skin, ears, nose, eyes, taste buds) to the brain However, most materials cannot move freely across the cell membrane. The selective permeability of the cell membrane allows some materials to pass trough it, but prevent the movement of others. Materials may move across the plasma membrane in two ways: passive transport and active transport, with the name indicating the main difference between them: passive transport does not require energy expenditure by the cell, while active transport requires the use of energy provided in the form of ATP molecules. One common passive transport mechanisms is diffusion, molecules move naturally from an area of their higher concentration to an area of their lower concentration, molecules move then down a concentration gradient. Osmosis is a particular type of diffusion in which the molecules that move down a concentration gradient are water molecules. Facilitated diffusion is another passive mechanism: the movement of molecules down a concentration gradient but with the participation of carrier proteins located in the cell membranes that help molecules to move into and out of the cell. Active transport is the only mechanism that involves the use of energy and always occurs up a concentration gradient, as opposite to diffusion. The hotter the medium, the faster the molecules diffuse Molecules of liquids and gases are always in constant motion, and this motion is temperature dependent. The more heat we add to the molecules, the more energy they have and the faster they move, this type of energy of movement is referred as to kinetic energy. Temperature is a measure of the average kinetic energy of the particles in a sample of matter. In conclusion, the more heat we add to the medium the faster the molecules will move and diffuse and the higher the temperature will be. TASK 1: TEMPERATURE AND THE RATE OF DIFFUSION Procedure: DEMO: Your instructor will assign this task to one of the groups 1. Fill 2/3 of a 500ml beaker with water and add enough ice to bring the temperature of the water close to the freezing point (0ºC). Add a thermometer to the beaker to check the previous and wait until the temperature is about 4ºC. 2. Fill 2/3 of another 500ml beaker with hot water, check the temperature of the water with a thermometer and be sure is above 50ºC. 3. Removed the ice from the beaker and place the two beakers on sheets of white paper and record the temperature of each before proceeding to the next step. TEMPERATURE BEAKER 1: ºC, TEMPERATURE BEAKER 2:.ºC 4. Add a grain of potassium permanganate to each beaker 5. Observe what happens in the two beakers over a five (5) minutes period General Biology 2 Instructor: Jose Bava, Ph.D

1) What are the independent (X) and dependent (Y) variables we used in this experiment? X=... Y=... 2) In what beaker the molecules of potassium permanganate diffused faster?.. 3) According to the results, what is the relationship between temperature and diffusion? The lighter the molecule, the faster it diffuses It seems common sense to think that the heavier an object is, the harder it will be to move it from one place to another. When it comes to molecules, we find that the same principle applies: big and heavy molecules will diffuse much more slowly than small and light molecules. This is the hypothesis we will test with the following experiment. TASK 2: MOLECULAR WEIGHT AND THE RATE OF DIFFUSION Procedure: 1. With a cork borer, make two holes in separate parts of a petri dish with agar gel, as indicated in the figure on the right. 2. Fill two holes with potassium permanganate and two holes with methylene blue 3. Refill the holes after 15 minutes 4. Wait 45 minutes more and determine the rate of diffusion by measuring the diameter of each colored circle in the agar plate 5. Write in the following table the results obtained and graph your results in the space provided Molecular weight Rate of diffusion (mm/h) Potassium permanganate 158 Methylene blue 374 4) What are the independent (X) and dependent (Y) variables we used in this experiment? X=... Y=... 5) What substance diffused more? 6) Make a bar graph showing your results 7) According to the results, what is the relationship between molecular size and diffusion?.. DIFFUSION RATE (mm)....... MOLECULAR SIZE General Biology 3 Instructor: Jose Bava, Ph.D

The net rate of diffusion depends on the concentration gradient So far, we have seen that higher temperatures increase the rate of diffusion and also that small molecules will naturally diffuse faster than large ones. How would the number of molecules in a medium, or concentration, affect the rate of diffusion? Will molecules diffuse faster if the concentration on both sides of the cell membrane is large when compared to a small difference? In order to examine this we will use the diffusion of water: osmosis. As any other molecule, water moves across the membrane with a net movement from where is more concentrated to where it is less concentrated. The concentration of water is inversely proportional to the solutes dissolved in it. In other words, water will always have a net diffusion in the opposite direction the solute molecules would try to move. TASK 3: OSMOSIS, EFFECT OF CONCENTRATION ON THE RATE OF DIFFUSION Procedure: DEMO: Your instructor will assign this task to one of the groups: Your lab instructor will setup two osmometers with different concentrations of sucrose or molasses (see figure). The cellulose membrane is not permeable to sucrose or molasses, so this molecule cannot diffuse out of the bag. As a consequence of the previous, osmosis will happen: the molecules of water in the beakers will diffuse across the membrane and enter the bags in order to balance the concentrations. As water accumulates in the bags the only place it has to go is up the glass tubes The hypothesis is that osmosis will happen at a faster rate where the sucrose is more concentrated. The instructor will mark the level of the fluid at the beginning and at four different times and you will estimate the rate of diffusion. 8) What are the independent (X) and dependent (Y) variables we used in this experiment? X=... Y=... 9) Keep track of the osmosis rate every 30 and record the information in the table provided below Real Time (hour) Start:... Cumulative time (Min) 0 Osmosis in 50% Molasses (cm) Osmosis in 90% Molasses (cm) 30 60 90 120 10) In what system the molecules of water diffused faster?. General Biology 4 Instructor: Jose Bava, Ph.D

11) Make a line graph showing your results for both concentrations. Use a solid line for 90% molasses and a dashed line for 50% molasses 12) According to the results, what is the relationship between concentration and diffusion? OSMOSIS RATE (ml) 0 30 60 TIME (min) 90 120 Molecules diffuse across membranes depending on membrane permeability We mentioned before that cells have a selective membrane; the membrane is not permeable to all molecules that move into and out of the cell. Materials that have a small molecular size will diffuse across the membrane more easily than bigger ones, with large molecules being prevented from passing through. This selectivity of the cell membrane can be explored with a cellulose membrane (dialysis tubing) acting as a cell membrane. This cellulose membrane has many microscopic pores and some molecules will be able to diffuse using these pores, while some others will be too big and will be prevented from passing through. TASK 4: MOLECULAR SIZE AND MEMBRANE PERMEABILITY Procedure: 1. Cut one piece of cellulose tubing around 12 cm long. Tie one end of the cellulose tubing piece, be sure that a liquid will not pass through that end 2. Fill one bag with 2/3 glucose solution and 1/3 starch solution and tie the other end of the bag with a piece of string 3. Fill half of a 250 ml beaker with water and add several droppers of lugol s iodine solution to the beaker until the color of the solution appears very light brown. 4. Rinse off the cellulose bag with water to eliminate any molecules that may remained stuck to the walls of the cellulose tubing 5. Place the bag inside the beaker and wait for 45 minutes for the substances to diffuse Testing for biological molecules 6. Lugol s iodine has a brownish color and reacts only with starch, giving a dark blue/black if starch is present. The iodine is present in the beaker already, and the starch is present in the bag, so the only thing you have to do is observe where the dark blue/black color appears 7. Benedict s reacts with simple (reducing) sugars, changing from blue (negative) to green-yellow-orange-red (positive) if simple reducing sugars are present. Fill a test tube with about 2cm of water from the beaker (that contains the bag) Add to the test tube about 2cm of Benedict s reagent Heat the tube in the beaker that is setup already in the hot plate for 3 minutes and observe the change in color according to the scale given above General Biology 5 Instructor: Jose Bava, Ph.D

1) Summarize the results in the following table Test BAG BEAKER BEAKER Lugol s Iodine (Starch) Lugol s Iodine (Starch) Benedict s (Sugars) Result and final color ( + ) ( - ). ( + ) ( - ).... ( + ) ( - )..... 2) Did the starch diffuse across the cellulose membrane? YES / NO 3) Explain how you deduced the previous based on the chemical tests performed 4) Did the iodine diffuse across the cellulose membrane? YES / NO 5) Explain how you deduced the previous based on the chemical tests performed 6) Did the glucose molecules diffuse through the bag s wall? YES / NO 7) Explain how you deduced the previous based on the chemical tests performed 8) The circle given below represents the relative size of the membrane s pores; summarize your conclusions writing the names of the four molecules (water, iodine, starch, glucose) in the following size scale. The water is already provided for you as an example 9) How could you test if the water actually did osmosis into the bag? General Biology 6 Instructor: Jose Bava, Ph.D

Plant Cells and Osmosis Plant cells have three main differences with animal cells: the chloroplasts for photosynthesis, the cell wall for support and protection of the cell, and the central vacuole, a large, water-filled organelle that stores toxins, degrades enzymes, disposes of wastes, and is involved in cell growth. When well watered, plant cells increase the size of the central vacuoles and this exerts pressure, pushing all the other organelles against the inside of the cell wall. This pressure from the central vacuole supports the shape of leaves and other parts of the plant. Water loss causes the plant to wilt. What will happen to cells if an unusual salt concentration is placed outside the cell? The inside of the cell will have much less salt than the outside, Tonicity Tonicity is the relative concentration of solute outside the cell compared to that inside the cell. Solutes are the particles dissolved in the solvent, like glucose, sodium, etc.), which normally is a watery medium in living things. Useful terms when comparing solute concentrations on both sides of the membrane are: Isotonic: An isotonic cell has the same concentration of solute (salts, sugars, etc) and water as the solution outside the cell. If you place cells in an isotonic solution, no net transport across the membrane will take place, the shape and size of the cell will not change. Hypotonic: If the cell is hypotonic compared to the solution outside, then the cell has less solute dissolved (but more water) than the solution outside. If the solute in the solution outside cannot diffuse inside of the cell, then water from the cell will diffuse out to try to dilute the solute outside; the cell will shrink as a result. Hypertonic: When the cell is hypertonic, the cell has more solute dissolved (but less water) than the solution outside. If the solute inside the cell cannot diffuse out, then water will mainly diffuse inside the cell trying to dilute the solute inside, the cell will then swell or inflate. TASK 5: POTATO CELLS AND OSMOSIS Procedure: DEMO: Your instructor will assign this task to one of the groups 1. Cut two (2) strips of potato, each about 1cm wide and at least 5 cm long. Make sure both strips are have the same length. Record the original length in the table below. Potato cells have a salt concentration of about 0.9% inside and the salt stays constant, will not diffuse in or out as the situation changes in the outside, but water will! 2. Put one potato strip in a test tube labeled with 10% SALT WATER. Add enough 10% salt solution as to cover the potato strip 3. Put one potato strip in a test tube labeled with DISTILLED WATER. Add enough distilled water (0% salt concentration) as to cover the potato strip 4. Leave both tubes in a rack for one hour and then place the potato strips on a paper towel. Measure the length of each one and record the results in the table below 5. Touch and gently squeeze the potato strips and answer the following questions Potato strip in SALT WATER Potato strip in DISTILLED WATER Original length Final length Difference 10) Which one of the strips is stiff? What happened to those potato cells? Explain General Biology 7 Instructor: Jose Bava, Ph.D

11) Which one of the strips is limp, flexible? What happened to those potato cells? Explain............ 12) Which one of the solutions in the tubes is hypotonic to the cells, which one is hypertonic?...... LENGTH (cm) 8 7 6 5 4 3 2 13) Make a bar graph showing your results in the space provided, use two bars for each potato strip to show the strip s size at the beginning and at the end of the experiment TASK 6: ELODEA CELLS AND OSMOSIS 1 Initial Final Initial Final 0% (distilled water) 10% (saltwater) When well watered, elodea cells increase the size of the central vacuole and this exerts pressure, pushing all the other organelles against the inside of the cell wall. The cell appears under the microscope filled with chloroplasts occupying the space between the central vacuole and the cell wall. When cells lose water, the central vacuole and the cytoplasm, including the chloroplasts, pulls away from the cell wall Procedure: 1. Label a microslide with the number 1. Prepare a wet mount of an elodea cell and add a drop of the water where the elodea sprigs are being kept before covering the leaf with a cover slip. Elodea cells have a salt concentration of approximately 0.9%, normal water has a concentration of about 1% 2. Label a microslide with the number 2. Add a drop of 10% salt solution (sodium chloride) to the slide and prepare a wet mount of an elodea leaf, add a cover slip 3. Label a microslide with the number 3. Add a drop of distilled water (0% salt concentration) to the slide and prepare a wet mount of an elodea leaf, add a cover slip 4. Observe the slide 1 under the microscope and sketch the chloroplasts distribution it in the space provided below 5. Wait five minutes before observing slides 2 and 3 6. Observe the slide labeled 2 and sketch the chloroplasts distribution it in the space provided below 7. Observe the slide labeled 3 and sketch the chloroplasts distribution it in the space provided below Normal Water (1%) Saltwater (10%) Distilled water (0%) General Biology 8 Instructor: Jose Bava, Ph.D

14) Complete the following table with the missing information (see procedure for concentration info) SLIDE 1 SLIDE 2 SLIDE 3 salt concentration in Elodea cells 0.9% 0.9% 0.9% salt concentration in Water outside Tonicity of elodea cells (hypo, hyper, iso) 15) In which one of the slides the elodea cells swelled, with the central vacuole exerting pressure against the cell wall? Explain what happened for the cells to change their shape in that way 16) In which one of the slides the elodea cells shrink because the central vacuole is reduced in size? Explain what happened for the cells to change their shape in that way 17) In which one of the slides the elodea cells are most likely isomostic with the medium outside? Summary 18) Compare the different transport mechanisms by completing the following information DIFFUSION OSMOSIS ACTIVE TRANSPORT Occurs when gradient is? (pick the right option) High to Low High to Low High to Low Low to High Low to High Low to High Is it passive or active? Is a membrane is needed? Energy is needed? Energy, if needed, is provided by General Biology 9 Instructor: Jose Bava, Ph.D