BIOL 1406 OSMOSIS OBJECTIVES: 1. Define solvent, solute and solution. 2. Define osmosis. 3. Define isotonic, hypotonic, and hypertonic as they relate to relative concentrations of osmotically active substances. 4. Determine the direction and rate of osmosis into and out of simulated cells. 5. Explain why diffusion and osmosis are important to cells. 6. Describe how a isotonic, hypotonic and hypertonic solution affects plant cells. MATERIALS: ITEM NUMBER Pre-soaked dialysis tubing 15 cm long 2 per group; 4 per group String or dialysis clips 4 per group; 8 per group Graduated cylinder Phenolphthalein 1 bottle per group Starch suspension 150 ml per class 250 ml beaker 2 per group; 4 per group Iodine 1 bottle per group 1 M Sodium Hydroxide 1 bottle per group Sharpie Electronic balance Weigh boat Clock 1 per class 1% sucrose 1000 ml per class 10% sucrose 1000 ml per class 25% sucrose 1000 ml per class Elodea leaf Blank slides Coverslip 30% sodium chloride 1 bottle per table Distilled water 1 bottle per table INTRODUCTION: To maintain homeostasis cells must be able to regulate what enters and exits the cell. That job is assigned to the plasma membrane, which is selectively and differentially permeable. The cell membrane is a phospholipid bilayer (two layers) that surrounds the cell. Each layer consists of an outer phosphate head that is hydrophilic (water loving) and an inner lipid tail that is hydrophobic (water fearing). Molecules soluble in lipid and those that are non-polar are able to pass through the cell membrane. Phosphate heads Lipid tails Phosphate heads 1
Polar molecules have positively and negatively charged areas. Non-polar molecules do not have those charged areas on them. Molecules that are small and nonpolar pass through the cell membrane more readily than those that are large and polar. Molecules such as glucose, amino acids and charged ions can get across the membrane by passing through the hydrophobic portion of the membrane through hydrophilic areas called carriers. Small, hydrophobic molecules such as oxygen can readily pass through the cell membrane. Questions: 1. What kind of molecules would you expect to enter a human cell? 2. What kind of molecules would you expect to exit a human cell? We will use a substance called dialysis tubing to simulate a living cell. Dialysis is the diffusion of a solute across a membrane. Dialysis tubing is a good model of the cell membrane because it has small pores that allow water and small substances to move across the membrane. Unlike dialysis tubing, living cells also select for molecules based on their charge and solubility. Osmosis is the diffusion of water across a selectively or differentially permeable membrane from a region where it is highly concentrated to a region where it is in lower concentration. When describing the process of osmosis and dialysis, three terms are important to understand. 1. Solvent refers the to dissolving agent. 2. Solute refers to the dissolved substances. 3. Solution is the homogenous mixture of two or more kinds of molecules. Procedure 10.1A Diffusion across a differentially permeable membrane The following procedure will use two indicators: phenolphthalein and iodine. Phenolphthalein is a ph indicator that turns red in basic solutions (ph > 7) and is clear in acid solutions (ph < 7). Iodine changes from golden yellow to dark blue in the presence of starch. MATERIALS: ITEM NUMBER Pre-soaked dialysis tubing 15 cm long 2 String or dialysis clips 4 Graduated cylinder 1 Phenolphthalein 1 bottle Starch suspension 10 ml 250 ml beaker 2 Iodine 1 bottle 1 M Sodium Hydroxide 1 bottle Sharpie 1 1. Obtain 2 pieces of pre-soaked dialysis tubing and 4 pieces of string or dialysis clips. 2. Fold one end of each bag over 1-2 cm, then accordion-fold this end and tie it tightly with the string or clip. Be sure it is tied tightly because leaks will impact the outcome. 2
3. Roll the untied end of the bags between your thumb and finger to open the bag. 4. Fill one bag with 10 ml of water (use a graduated cylinder to measure this) and add 3 drops of phenolphthalein. 5. Seal the open end by the same method as in step #2. 6. Fill the other bag with 10 ml of starch suspension and seal the open end. 7. Rinse the outside of each bag gently with tap water. 8. Fill one 250 ml beaker with 200 ml of water and add 15 drops of 1 M sodium hydroxide. Number this beaker #1. 9. Place the bag containing phenolphthalein in the beaker. 10. Number the other beaker #2. Fill beaker #2 with 200 ml of tap water and add 20-40 drops of iodine. 11. Place the bag containing the starch suspension in beaker #2. 12. Observe the color changes in the bags and in the surrounding water. 13. Sketch and label the beakers and bags below. Record the color in each beaker and each bag. Questions: 1. Describe the color changes in each of the bags. 2. Which molecules passed through the bags (phenolphthalein, iodine, starch, sodium ions and/or hydroxide ions)? 3. What does the color change in beaker #1 indicate about the ph of the solution? Procedure 10.2B Osmosis through a selectively permeable membrane across a concentration gradient The following experiment will simulate osmosis across a concentration gradient. We sill use four dialysis bags that each contain an assigned concentration of sucrose solution. Bag A will contain a hypotonic solution. Hypotonic indicates that a given solution has a lower solute concentration than the solute concentration of its surrounding environment. Water moves across a selectively permeable membrane away from the hypotonic solution. Bag B 3
will contain an isotonic solution. When two solutions have equal concentrations of solutes, they are said to be isotonic. Water will continue to diffuse across the selectively permeable membrane into and out of the dialysis tubing at an equal rate. Bags C and D will contain hypertonic solutions. Hypertonic indicates that concentration of a given solution is greater than its surrounding environment. Water moves into the bag that is hypertonic. The sucrose solution does not move through the dialysis tubing. MATERIALS: ITEM NUMBER Pre-soaked dialysis tubing 15 cm long 4 per group String or dialysis clips 8 per group Graduated cylinders Sharpie 250 ml beaker Electronic balance Weigh boat Clock 1 per class 1% sucrose 1000 ml per class 10% sucrose 1000 ml per class 25% sucrose 1000 ml per class 1. Obtain 4 pieces of pre-soaked dialysis tubing and 8 pieces of string or dialysis clips. 2. Fold one end of each bag over 1-2 cm, then accordion-fold this end and tie it tightly with the string or clip. Be sure it is tied tightly because leaks will impact the outcome. 3. Roll the untied end of the bags between your thumb and finger to open the bag. 4. Fill the bags with the contents as indicated below. Label each beaker/bag with the appropriate letter A, B, C or D. 25% sucrose 1 % sucrose 1 % sucrose 1 % sucrose Bag A 10 ml of 1% sucrose Bag B 10 ml of 1% sucrose Bag C 10 ml of 10% sucrose Bag D 10 ml of 25% sucrose Beaker A Beaker B Beaker C Beaker D 5. Loosely fold the open end of each bag and carefully squeeze the excess air out. Fold and tie with the string/clip the open end of each bag. 6. Rinse each bag and check for leaks. 7. Blot each bag gently and weigh individually to the nearest 0.1 g. 8. Record the initial weight on the chart below. 9. Place Bag A containing 10 ml of 1% sucrose in Beaker A containing 150 ml of 25% sucrose solution. 4
10. Place Bag B containing 10 ml of 1% sucrose in Beaker B containing 150 ml of 1% sucrose solution. 11. Place Bag C containing 10 ml of 10% sucrose in Beaker C containing 150 ml of 1% sucrose solution. 12. Place Bag D containing 10 ml of 25% sucrose in Beaker D containing 150 ml of 1% sucrose solution. 13. Remove the bags at 15 minute intervals for one hour. Gently blot them dry and weight them to nearest 0.1g. Be careful with the bags to avoid leaks. Return them quickly to their containers. Chart 10.2B Bag A Bag B Bag C Bag D Changes of Dialysis Tubes 0 Min 15 Min 30 Min 45 Min 60 Min Initial Total Total Total Total Change in Questions: 1. In which bags did osmosis occur? Change in Change in Change in 2. Which bag represented the steepest concentration gradient? 3. Which beaker(s) of water represented a hypertonic environment? 4. Which beaker(s) of water represented a hypotonic environment? 5. Which beaker(s) of water represented a isotonic environment? Procedure 10.3 Graphing 1. Use the graph paper on the following page to record Total (g) vs. Time (min). The independent variable that you actively control is graphed on the X-axis (horizontal). The dependent variable is recorded on the Y-axis (vertical) and responds to differences in the independent variable. 2. Which is your independent variable, Total or Time? 3. Which is your dependent variable, Total or Time? 5
4. Graphs MUST have a title, correctly labeled axes and a label showing measurement units (e.g. min. or g), and values along each axis (e.g. 0, 15, 30, 45, 60). These should all be included on your graph. 5. Plot the data for total weight at each time interval on the graph at the end of the lab. 6. Include data for all 4 bags. 6
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Procedure 10.4 Osmosis in Plant Cells Plant cells contain a large fluid-filled central vacuole that allows the flow of water into and out of the cell. When a plant cells is placed in a hypotonic solution, water moves into to cell and the central vacuole expands. The cell wall restricts too much expansion resulting in turgor pressure (pressure of the plasma membrane on the cell wall). The high turgor pressure prevents further uptake of water by the cell. When a plant cell is placed in a hypertonic solution, water moves out of the central vacuole into the environment. The plasma membrane shrinks and pulls away from the cell wall. This process is called plasmolysis. MATERIALS: ITEM NUMBER Elodea leaf Blank slides Coverslip 30% sodium chloride 1 bottle per table Distilled water 1 bottle per table 1. Prepare a wet mount of a single layer of Elodea leaf. Examine the leaf at 4X, 10X and 40X. 2. Add 2 drops of 30% sodium chloride (NaCl) to the edge of the coverslip. 3. Wick the salt solution across the Elodea leaf. Allow the leaf to sit for 1 minute. 4. Examine the leaf at 40X. 5. Below sketch the leaf at 40X. 6. Now add distilled water to the edge of the coverslip. 7. Wick the solution across the Elodea leaf. Allow to sit for 1 minute. 8. Sketch the leaf below at 40X. Questions: 1. Describe the appearance of the Elodea leaf in the NaCl. 2. Describe the appearance of the Elodea leaf in the distilled water. Which Elodea leaf represented plasmolysis? turgor pressure?biol 1406 Assignment: Lab Report 8
Write the report over Osmosis Experiment (or can be modified for another exercise, e.g. enzyme lab) Use the following format: Title: (10 points) The title should clearly describe the experiment in 10 words or less. A title such as Osmosis is too general. Authors: (5 points) List your name first and last and the names of your lab partners. Your name should be listed first. Be sure to write down the correct spelling of your lab partners names. Abstract: (15 points) Indent the entire abstract. The abstract summarizes the experiment. It should include the following: (1) objectives and scope of the experiment, (2) summary of the methodology, (3) data summary, and (4) conclusions. NO REFERENCES HERE. Introduction: (15 points) This section includes the research done before starting an experiment. The introduction provides a description of the nature and background of the problem studied. The last part of the introduction states the objectives of your experiment. Each source of information must be cited. You must show the reference, even when you summarize someone s ideas and information. You may use your textbook and/or internet resources to provide the background information on osmosis. Since you worked with plant cells (carrots or potatoes) you can describe the effect of different types of solutions (hypertonic, hypotonic, and isotonic) on a plant cell. Place the name of the author and copyright year in parentheses at the end of the paragraph to show the source of your information. It is best to put the information in your own words, but you still give credit to the original source. If you copy information directly, use quotes and include the page number. Example: Osmosis is. (Campbell and Reece, 2005, p. 350) Materials and Methods: (15 points) Write this section in a paragraph format. Summarize the procedure and mention the important materials in your description. Describe how, when, and where you conducted the experiment. Include the equipment, chemicals, and materials used. You must include enough detail so that another scientist can replicate your work. Results: (15 points) Summarize what happened in one or two sentences. Attach a data table and graph. You may incorporate the data table into the results or say See table 1 and place the table at the end of the report. Lab 4 Water, gives directions for creating a line graph using the program Excel. The independent variable is the variable that the scientist manipulated during the experiment. The independent variable will be the Time (in different sucrose solutions). Plot Time (Minutes) on the x-axis. It is shown on the x-axis of the graph. The dependent variable changes in response to the changes in the independent variable and is shown on the y-axis. The dependent variable is Total (g). Every graph should have a title. The x-axis and y-axis should be clearly labeled including the units. You can draw lines for the four different concentration gradients on the same graph. Discussion: (20 points) What do the results mean? You must explain the data for each sucrose solution used. Explain any unexpected results. Did the results match your hypothesis? Explain why the weight changed or did not change for each solution. If the results are unclear from your data, recommend that further testing be conducted. Include any recommendations for the experiment. (Recommend that distilled water be used next time.) 9
References: (5 points) Use APA format. Format for non-periodicals (books) Example: Campbell, N. A., & Reece, J. B. (2005). Membrane structure and function. Biology (pp. 124-140). San Francisco: Pearson Benjamin Cummings. Authors, copyright date, chapter title, book title, page numbers, publication city, publisher. APA format: Website Example: Hutchings, S. (2006). Osmosis using plant cells. Retrieved Feb 28, 2007 from http://hutchings.com Authors, (copyright date, if not given put n.d.). Title of website. Retrieved (month, date, year) from http://website address 10
Section Elements Comments Total Points Title (10 pts) 10 words or less Includes: sucrose, concentration gradient (different solutions), dialysis tubing, weight (mass) Authors (5 pts) First then last name, including lab partners Introduction (15 pts) Complete sentences Sources cited Osmosis Tonicity (Hypertonic ) Dialysis Tubing Materials and Methods (15 pts) Results (15 pts) Discussion (20 pts) References (5 pts) Typed Paragraph format Include: dialysis tubing, weight, sucrose solution description Includes all bags in description Someone could replicate the work based on this section Summary sentence(s) to describe data Data table attached Graph: with the following elements Title x-axis labeled with time y-axis labeled with weight All bags shown on graph(s) Appropriate units Explains the meaning of the results for each bag (A, B, C, and D) Explain why the weights changed Compare the results for C and D and explain the difference in weight gain Unexpected results explained Listed in APA format 3. 11