Lab Copy: Movement Across a Membrane PDF

LABORATORY EXERCISE 7 Movement Across a Membrane Diffusion and Osmosis Learning Objectives: *Understand the important concepts relating to diffusion of gasses. *Understand how concentration affects movement across a membrane. *Explain how movement across a membrane is affected by surface area to v...

LABORATORY EXERCISE 7 Movement Across a Membrane Diffusion and Osmosis Learning Objectives: *Understand the important concepts relating to diffusion of gasses. *Understand how concentration affects movement across a membrane. *Explain how movement across a membrane is affected by surface area to volume ratios. *Apply principles of diffusion to cell structure and size A cell’s plasma membrane protects a cell. It provides a place where the cell’s cytoskeleton can attach, and thereby help to maintain a cell’s shape. The plasma membrane is also selectively semi-permeable. Movement across a cell’s plasma membrane can occur two ways. 1. Passive transport (requires no cellular energy). Types of passive transport are: diffusion, osmosis, facilitated diffusion. 2. Active transport (requires cellular energy). Types of active transport are: Primary active transport using ATP and Secondary Active transport using an electrochemical gradient. Facilitated diffusion and forms of active transport all share the common aspect that they are mediated through transmembrane proteins. In today’s exercise you will be observing two types of passive transport that occur in all living cells diffusion and osmosis. Diffusion and osmosis play an important role in a cell’s ability to maintain homeostasis. However, there is a limit to the utility of passive transport in a living cell. In the final exercise you will observe one of passive transport’s limitations. You will observe the restrictive effect on cell size that is influenced by surface area to volume ratios. Diffusion The molecules of all materials (solid, liquid and gas) tend to diffuse "randomly" due to molecular motion (i.e. heat) and the natural increase in entropy. On average, this "moves them" from regions where they are more concentrated to regions where they are less concentrated. For solids, especially for crystalline solids, diffusion is quite slow. In liquids, diffusion is faster; in gases it may be faster still. Qualitative versus quantitative observations With any scientific procedure you could make both quantitative and qualitative observations. It is important that you can determine if the observations you are making are quantitative or qualitative and the advantages and disadvantages of each type of observation. When making qualitative observations you will collect numerical data and then evaluate the data (using statistical analysis). This data could be used to support or reject a hypothesis. Quantitative observations use non-numerical observations and are subjective to an individual’s observations. By incorporating both observations in your lab, you can better understand the reaction. An example of the two types of observations would be: When observing the candle burning, a quantitate observation would be “the flame is hot”, and a qualitative observation would be “the temperature of the flame is 200 degrees Celsius”. Balloon Demonstration (effects of a differentially permeable membrane). Note: There are 2 different procedures that can be used for this demonstration. Your instructor will choose which demonstration to be performed in your lab. If you choose procedure 1 you will be using quantitative observations. If you choose procedure 2 you will be using qualitative observations. If you are using procedure 1, your instructor will provide you with qualitative data. Procedure 1: Observation of the balloons to determine if air or CO2 moves across the balloon membrane. a. Take four identical rubber balloons. b. Fill two with CO2 from a gas cylinder. It is important that each is the same volume. Make sure you can fit them into the jars. c. Fill two with air (you are the source or use air in fume hood). It is important that each is the same volume. Make sure you can fit them into the jars. d. Put each balloon into a separate wide-mouth jar. Flush two of the jars with CO2 (it's heavier than air so the air will be displaced up and out) and two of the jars will be filled with the air from the room. Use the table below to ensure the combinations of balloon-to-jars are the same as the table: e. Make your initial observations on Table 6.1 Table 7.1. Balloon data Contents of Balloon Contents of Jar Initial observation Final observation AIR AIR _____________ _____________ AIR CO2 _____________ _____________ CO2 AIR _____________ _____________ CO2 CO2 _____________ _____________ After 2 hours, record your observations of the balloons in Table 7.1 Questions to consider as you look at the results: What happened? Why does it happen? Is the thin rubber balloon more permeable to carbon dioxide or air? Procedure 2: Observation and measurement of the balloons to determine if air or CO2 moves across the membrane of the balloon. You will be following the same setup that is used in Table 6.1 above, however, you will be using a measurement of the balloon’s volume in addition to your visual observations. a. Place a battery jar in a grey plastic tub and fill the jar to the top with water (fill it to the brim but not to the point of overflowing into the tub) b. Take out four identical rubber balloons. c. Fill one balloon with air (make sure it has room to fit into the open jar) and tie off the end. d. Holding the end of the balloon with your thumb and index finger push the balloon under water that is in the battery jar (you may need to use a third finger to prevent the balloon from moving and popping up). Remember how you hold the balloon (you do not want different volumes due to your fingers being under water differently; this could affect your volume measurement). e. Remove the balloon, dry it with a towel, and place it in the jar and close the lid. Record this time f. Take the battery jar out of the tub. Using the funnel and graduated cylinder pour the water that is in the grey tub into the graduated cylinder. Measure and record the initial volume on Table 7.2. g. Place the battery jar back into the grey tub and pour the water that is in the graduated cylinder back into the battery jar (this should refill the jar). h. You are going to repeat the steps d to g for the other 3 balloons; you will be following Table 6.2 below when filling the balloon and jar with air or CO2. Putting each balloon into a separate wide-mouth jar, flush two of the jars with CO2 (it's heavier than air so the air will be displaced up and out) and two of the jars will be filled with the air from the room. Table 7.2 Balloon data Contents of Balloon Contents of Jar Initial volume Final volume Change in volume Air Air _____________ _____________ _____________ Air CO2 _____________ _____________ _____________ CO2 Air _____________ _____________ _____________ CO2 CO2 _____________ _____________ _____________ After 2 hours (Note: each jar will have a different start time and thus a different end time) use the same technique to measure the volume of the balloon and record it in Table 7.2. The size of the balloons can now be quantified with this water displacement technique. Questions to consider as you look at the results: What happened? Why does it happen? Was there a difference in your visual observations and the observations that were measured? Is the thin rubber balloon more permeable to carbon dioxide or air? Osmosis Osmosis is specifically defined as the diffusion of water across a differentially permeable membrane, under conditions where solute molecules (i.e. dissolved molecules) are unable to diffuse. Membranes that let certain molecules pass through them, while stopping other molecules, are called "differentially permeable" membranes. There are many examples of differentially permeable membranes. The rubber balloon is a differentially permeable membrane. The differentially permeable membranes of cells are made of phospholipids and proteins. In this exercise a piece of dialysis tubing will serve as a membrane. Water molecules pass easily through a cellulose membrane (dialysis tubing). This is because the “holes” or “spaces” between the cellulose molecules are large enough to allow passage of the small water molecule. However, sucrose molecules do not cross the cellulose membrane. When water passes through a differentially permeable membrane by diffusion, and sucrose dissolved in the water cannot pass through the same membrane, osmosis has occurred. Procedure a. Select a piece of dialysis tubing about 20 cm long, which has been soaked in water. Fold downward approximately 4 cm of one end of the dialysis tubing. Fold the same end a second time (about 1.5 cm) and place a binder clamp across the double-folded region. b. Turn the unclamped end upwards and open the dialysis tubing. Fill the dialysis tubing with the sugar solution so it is 1/3–full. Double-fold and clamp the open end making certain to leave an air space in the dialysis tubing that measures at least 3-4 cm. c. Blot dry the tubing/clamp set-up. Weigh the set-up by placing it on the scale. Record the weight in Table 6.3. d. Fill the large, tall beaker with DI water. Immerse the dialysis tubing set-up in the water. Allow osmosis to occur for five minutes. e. Remove the dialysis tubing. Gently blot the tubing dry. Obtain a final weight and record it in Table 6.3. Your instructor may want you to continue to measure the tubing every 5 minutes. Check with your instructor! f. Repeat the above steps for each sugar solution. Table 6.3 10% sugar initial weight ________; final weight ________ change in weight ______ 20% sugar initial weight ________; final weight ________ change in weight ______ 30% sugar initial weight ________; final weight ________ change in weight ______ Explain why there are differences in the rates of osmosis (change in weight over time) when comparing the three different sugar solutions. Diffusion rates depend on a cell’s surface-to-volume ratio The upper size limit of a cell is partially determined by the time required for diffusion to distribute nutrients and remove waste. This part of the exercise will emphasize how diffusion times depend on surface-to-volume ratio. Measure the diffusion rate of sodium hydroxide (NaOH) through four agar cubes of different size. Because NaOH is colorless, an indicator dye called phenolphthalein will be used to help track its movement. Phenolphthalein turns bright pink when it reacts with NaOH. Procedure 1. Obtain pieces of agar from your instructor. Use a knife to CAREFULLY cut the agar into blocks (cubes) of the following sizes: 0.5 cm, 1.0 cm, 2 cm, and 3 cm. Please do not be wasteful of the agar. 2. Calculate the surface area, volume, and surface-to-volume ratio of each cube. (Area = 6 s2; Volume = s3; where s is the length of one side of a cube). Enter your data in Table 6.4 below. 3. Place all four agar cubes in a finger bowl so that they are separated and not touching. Fill the bowl with enough 0.01M NaOH to completely cover them. 4. With a plastic or wooden spoon or probe, turn the cubes frequently to allow for even diffusion at all sides. Be careful not to break or damage the cubes. 5. Allow 12 minutes for the diffusion process. Note: Your instructor may vary this time. Check with your instructor for the exact time. 6. Gently carry the finger bowl to a sink and pour the NaOH down the sink. Be careful to not lose any of the blocks. Fill the finger bowl with water 3-4 times to gently rinse the agar blocks. Remove the blocks and place them on a paper towel to drain. 7. Quickly make a slice through the center of each cube. Make a second slice of ~2-3 mm depth from the center edge. Quickly measure the distance (to the nearest mm) where diffusion of NaOH has NOT taken place. 8. Record your data in the Table below (the column labeled s'). The volume occupied by the NaOH can now be obtained by subtracting the volume of the interior cube [s'3] from the volume of the entire block [s3]). Figure 6.2. This diagram is what you would have seen when you cut the cube in half. Table 6.4 Surface-to-volume calculations: diffusion of NaOH into agar/phenolphthalein blocks Cube# Side(s) size. Surface Area (cm2) 1 0.5 cm ________ _______ _____________ 2 1.0 cm ________ _______ _____________ 3 2.0 cm ________ _______ _____________ 4 3.0 cm ________ _______ _____________ Cube#. s’ 1 Volume (cm3)s3 Surface area to Volume ratio s’3 ______ Volume with NaOH s3 - s’3. ________ _______ Ratio of diffused area s3 - s’3. / s3 _____________ 2 ______ ________ _______ _____________ 3 ______ ________ _______ _____________ 4 ______ ________ _______ _____________ Fill in the above spaces with your observations/results Determine the following relationships: 1. How does the surface-to-volume ratio of a cube relate to its size(s)? 2. How does the volume of diffused NaOH depend on the surface-to-volume ratio of a cube? DATA SHEET for DIFFUSION AND OSMOSIS: EXERCISE 6 Name__________ Turn in the following questions, these questions will be graded 1. BALLOON DEMONSTRATION A. Explain what happened with the diffusion of air/carbon dioxide in the balloons Air in the Balloon---CO2 in the jar: CO2 in the Balloon---Air in the jar: CO2 in the Balloon--- CO2 in the jar: Air in the Balloon---Air in the jar: B. Is rubber balloon more permeable to carbon dioxide than to nitrogen or oxygen (i.e. air)? C. The “Air in the Balloon---Air in the jar” and “CO2 in the Balloon--- CO2 in the jar” have the same gases on each side of the balloon. Was there a difference in the observation of these balloons? D. Do you think the elasticity of the balloon membrane applied pressure on the gas within to significantly affect the gas’s diffusion across the membrane? If not, why? 2. OSMOSIS A. Record your measured rates (in grams per minute) of osmosis for: 10% sucrose: g/minute 20% sucrose: g/minute 30% sucrose g/minute B. Explain why the rates of osmosis were different: C. What would happen to the tubes if left in the beaker for 2 hours? 3. DIFFUSION RATES DEPEND ON A CELL'S SURFACE-TO-VOLUME RATIO A. What substance diffused into the agar block? B. How does relative diffusion volume depend on the surface-to-volume ratio? C. Why is surface-to-volume ratio important for cells? D. Generate a hypothesis to explain why cells grow more rapidly immediately after cell division than after they have grown to a larger size. E. Speculate on how would you expect temperature to affect diffusion? F. What is the largest cell? Why do you think the cells of our body do not get this big?