Carbon Dioxide Uptake and Production in Photoautotrophic Elodea and Heterotrophic Goldfish BC1001 Revolutionary Concepts in Biology LAB 3, September 27-October 1, 2004 This week in lab you will use the scientific inquiry method to design and test a hypothesis. In lecture you have learned about autotrophic and heterotrophic organisms; in this lab you will measure aerobic respiration and photosynthesis. You will use a goldfish as an example of a heterotroph and Elodea as an example of an autotroph (the same aquatic plant whose leaf cells you examined under the microscope in lab 2). Please remember that while only the plant carries out photosynthesis, BOTH the PLANT and the ANIMAL carry out aerobic respiration in their mitochondria. Summary equation for aerobic respiration (animals and plants) C6H12O6 + 6 O2 6 CO2 + 6 H2O + ATP + Heat Summary equation for photosynthesis (plants) 6 CO2 + 6 H2O + light energy C6H12O6 + 6 O2 + Heat Note that carbon dioxide is produced by aerobic respiration and consumed by photosynthesis. In this experiment, you will be indirectly measuring amounts of CO2 in an aquatic system— allowing you to monitor respiration and photosynthesis. The goldfish will be producing CO2 through aerobic respiration, but the Elodea will be both producing CO2 through aerobic respiration and consuming CO2 through photosynthesis. CO2 reacts with water to form carbonic acid through the following reaction: CO2 + H2O H2CO3 In your experiments in lab this week, you will use a pH indicator (phenolphthalein) to detect changes in pH resulting from the production and consumption of carbonic acid (and thus CO2). Phenolphthalein is colorless in acidic solutions and pink in basic solutions. You will measure the volume of NaOH necessary to neutralize the carbonic acid produced by CO2; this is an indirect method that allows you to calculate the amount of CO2 produced or consumed. Lab 3-1 Scientific Inquiry Method Scientists use the scientific inquiry method to further scientific knowledge. The steps in this method are the following: make observations ask question develop hypothesis make prediction if results do not support the hypothesis, revise hypothesis or develop a new one design experiment collect data if results do support the hypothesis, make more predictions and test them analyze data draw conclusions The scientific method begins with the process of making observations. From these observations, you ask a question about what you see. Next, you develop a hypothesis, often defined as an educated guess to answer that question. You can make a prediction based on whether or not your hypothesis is true, and then design and carry out an experiment to test your hypothesis and prediction. Data (remember that “data” is plural, “datum” is singular) must be collected and analyzed. From the results of your experiment, you can draw conclusions about whether your hypothesis is or is not supported. You will use all the steps in the scientific method in lab this week. Please note that you can NEVER “prove” a hypothesis to be true, you can only have your hypothesis supported by data. NEVER, EVER use the word “prove” in a scientific setting. Really, NEVER. Lab 3-2 Preliminary experiments AUTOTROPHS VS. HETEROTROPHS Living things obtain their energy in a variety of ways. Some organisms are able to produce energy from sunlight or other environmental sources. Of the organisms that produce their own energy, the autotrophs, you are probably most familiar with the green plants. Other organisms, heterotrophs, cannot produce their own energy; they must obtain it from other organisms. There are several ways of making a living as a heterotroph. You can be an herbivore and obtain nutrients by eating living plant matter. You can be a carnivore and obtain energy by eating other animals. Or, you can be a saprophyte and obtain nutrients from decaying organic matter, as mushrooms do. Other organisms are parasites, obtaining energy from a plant or animal without, at least initially, killing it. Whether an organism is an autotroph or a heterotroph, one can get an idea of the rate at which it is carrying out metabolic processes by measuring the amount of carbon dioxide it uses or produces, respectively. Autotrophs convert carbon dioxide to oxygen and carbohydrates by photosynthesis. Both autotrophs and heterotrophs produce carbon dioxide as a result of metabolizing food in the presence of oxygen. In today's lab you will measure carbon dioxide use by a green plant and carbon dioxide production by an aquatic animal. While doing this exercise, think about how carbon dioxide and oxygen are involved in the lives of the organisms. In particular, keep in mind that autotrophs not only produce energy, they also use it. Textbook reading prior to coming to lab: Tobin and Dusheck 3rd edition: pp. 109-144. Lab 3-3 The following is a brief outline of the steps you will use to calculate carbon dioxide production/consumption by a heterotroph and an autotroph: H2CO3 CO2 H2CO3 CO2 Add phenolphthalein Add NaOH Collect data Analyze data Etc. Lab 3-4 A. CO2 UPTAKE AND PRODUCTION BY AN AQUATIC PLANT Natural waters such as ponds, lakes, and rivers are generally rich in bicarbonates [usually Ca(HCO3)2] that serve as a source of carbon dioxide for photosynthesizing aquatic plants. Using a relatively simple quantitative procedure, we can determine the uptake and production of CO2 by aquatic plants. Here, we will use Elodea, an aquatic plant commonly found in ponds and small lakes (remember from last week that Elodea is NOT an alga). Because plants both consume and produce carbon dioxide, you can only measure the NET carbon dioxide change. If there is an overall DECREASE in carbon dioxide, you know that more photosynthesis has occurred than aerobic respiration. If there is an overall INCREASE in carbon dioxide, you know that more aerobic respiration has occurred than photosynthesis. PROCEDURE: The italicized questions are for you to think about as your prepare for and carry out lab. Some of these might show up on your pre-lab quiz or on your exam. 1. Add 150 ml of spring water to a clean beaker. Using a clean straw, blow bubbles into the water for one minute total (this can be done over several shorter time periods) so that the water will be saturated with CO2. Why is it important that you blow bubbles into the total 150 mL of water instead of blowing separately into each 50 mL aliquot of water in the three beakers? 2. Divide the 150 mL of CO2-saturated water into three equal portions in three beakers. Label one beaker “Elodea, Light”, one beaker “Elodea, Dark”, and one beaker “No Elodea.” Why are you using a beaker with no Elodea? Think back to the first week and what you learned about the importance of controls in designing a good experiment. 3. Using forceps, obtain a piece of Elodea that is approximately 2 g in weight. Touch the plant briefly to a piece of paper towel to remove excess water (you do not need to dry it completely, just remove the biggest drops of water) and weigh it in a dish on the balance. Submerge the piece of Elodea in one of the beakers. Obtain a second specimen of the same weight for the other beaker (if your second piece is not the same weight, remove small pieces from the cut end until it is the same weight). The third beaker will have no plant sample. Why is it important to have the same amount of plant tissue in each of the experimental beakers? 4. Place the “Elodea, Light” beaker 12 inches away from a lamp with a 100-watt bulb. Place the “Elodea, Dark” beaker in the dark. Record the time you begin the experiment on Data Sheet I. Should you place your “No Elodea” beaker in the light or the dark? If it doesn’t matter, why doesn’t it matter? 5. At the end of 60 minutes, remove both pieces of plant and place them in the tank marked “Used Elodea.” Record the ending time on Data Sheet I. Save the water. Yes, this is a long time to wait. During the wait, you should begin your goldfish experiment and design your own experiment. Lab 3-5 6. Add three drops of phenolphthalein to each beaker. [Your “Elodea, light” beaker may already look pink after the addition of the phenolphthalein. This is OK, just add enough NaOH to the other two beakers to get the SAME shade of pink.] Phenolphthalein is a pH indicator that changes from colorless to pink as the solution changes from acidic to basic. The number of drops of NaOH needed to neutralize the solution so that phenolphthalein turns pink (basic) is a direct function of the acidity of the water. The more acidic (more carbonic acid / CO2) the solution is, the greater the number of drops of NaOH needed to obtain a color change. 7. Place your beakers on a sheet of blank white paper to help you see the colors more easily. With a dropper (the dropper delivers 0.05 ml per drop), slowly add 0.02 N NaOH to the “Elodea, Light” beaker, counting the number of drops until a faint but permanent pink color is obtained (if your beaker was already a faint, permanent pink color, do not add additional NaOH). Thoroughly stir the solution with a glass stirring-rod after adding each drop. 8. Count the number of drops of NaOH you need to add to the “Elodea, Dark” beaker to achieve the SAME pink color as in the “Elodea, Light” beaker. Record your results on Data Sheet I. 9. Count the number of drops of NaOH you need to add to the “No Elodea” beaker to achieve the SAME pink color as in the “Elodea, Light” and “Elodea, Dark” beakers. Record your results on Data Sheet I. 10. Use Table 3-1 to calculate the amount of CO2 consumed or produced in each of the beakers during the experiment. Record these amounts on your data sheet. Be sure to specify whether there was a net increase or net decrease in the amount of CO2 (in other words, did the use more CO2 in photosynthesis than it produced by respiration or did the plant give off more CO2 from respiration than it used in photosynthesis?). Lab 3-6 B. CO2 PRODUCTION BY AN AQUATIC ANIMAL To calculate the oxygen consumption of an aquatic animal, we can measure the amount of CO2 it produces in the water in a given period of time. We will use a method similar to that used in determining the CO2 uptake and production by Elodea. Note that the results of this experiment will be easier to understand because animals do not carry out both photosynthesis and respiration, only respiration. PROCEDURE: 1. Add 200 mL spring water to each of two clean beakers. Label one beaker “Fish” and the other beaker “No fish”. 2. Use the net to carefully and respectfully move a goldfish into the “Fish” beaker. The “No fish” beaker serves as the control. Again, why do you need to use a control in this experiment? 3. Note the time on Data Sheet I and leave the beakers on the table for 30 minutes. Every 5 minutes, make note of the fish's behavior. Is it quiet or does it swim around a lot? 4. At the end of 30 minutes, carefully remove the animal and return it to the aquarium. Record the ending time on Data Sheet II. 5. Add five drops of phenolphthalein solution to each beaker of spring water. 6. Place your beakers on a sheet of clean white paper to help you see the colors more easily. With a dropper (the droppers deliver 0.05 ml per drop), slowly add 0.02 N NaOH to the solution in the “No fish” control beaker until a faint, but permanent, pink color is obtained. Thoroughly stir the solution with a glass stirring-rod after adding each drop. Record your results in Data Sheet II. 7. Count the number of drops of NaOH needed to achieve the SAME permanent pink color in the “Fish” experimental beaker. 8. Using Table 3-1, calculate the amount of CO2 neutralized by the NaOH. The difference in number of drops of NaOH is an indirect measurement of the amount of CO2 produced by the goldfish. Lab 3-7 Table 3-1. Conversion Table of Drops of 0.02N NaOH to Volume of CO2 Values Difference in Number of 0.02N NaOH Drops 1 2 3 4 5 6 7 8 9 10 Volume of CO2 (mL) 0.05 mL 0.09 mL 0.14 mL 0.18 mL 0.23 mL 0.27 mL 0.32 mL 0.36 mL 0.41 mL 0.45 mL What happens if there is more than a 10-drop difference in the amount of NaOH used? You’ll need to come up with a mathematical formula, which shouldn’t be difficult to do. This lab was modified by Margaret Olney for BC1001 Revolutionary Concepts in Biology Lab, Barnard College, September 2003 and 2004. Lab 3-8 BIOLOGY 1001 NAME__________________________________ Lab day and time__________________________ Lab instructor_____________________________ LABORATORY 3: DATA SHEET 1 to be handed in at the beginning of next week’s lab Be sure to write out your answers to all questions (including the italicized ones) on the data sheets. DATA SHEET FOR PRELIMINARY EXPERIMENTS I. NET CO2 EXCHANGE BY AN AQUATIC PLANT A. Time experiment began: ________ Time experiment ended: ________ Length of experiment (in minutes): _________ B. Number of drops of NaOH added to “Elodea, light” beaker = ______ Number of drops of NaOH for “Elodea, dark” beaker = ______ Number of drops of NaOH for “No Elodea” = _____ C. Difference in number of drops for beaker in dark (compared to empty beaker) = ________ drops Difference in number of drops for beaker in light (compared to empty beaker) = ________ drops D. Amount of CO2 taken up or given off by plant in dark (from conversion Table 3-1) = _____ ml Here’s the hard part, you’ll have to think about whether this represents a net increase or net decrease in the amount of CO2—was more CO2 taken up than given off or more CO2 given off than taken up? _____________________________________________ Amount of CO2 taken up or given off by plant in light (from conversion Table 3-1) = _____ ml Here’s the hard part, you’ll have to think about whether this represents a net increase or net decrease in the amount of CO2—was more CO2 taken up than given off or more CO2 given off than taken up? _____________________________________________ Lab 3-9 E. Yes, you’ll have to do some math to figure these out. Amount of CO2 taken up or given off by plant in dark PER HOUR = _____ ml/hr net increase or decrease of CO2?__________________________________ Amount of CO2 taken up or given off by plant in light PER HOUR = _____ ml/hr net increase or decrease of CO2?__________________________________ II. CO2 RELEASE BY AN AQUATIC ANIMAL A. Time began: ________ Time ended: ________ Length of experiment (in minutes): _________ B. Number of drops of NaOH for “Fish” beaker = ______ Number of drops of NaOH for “No Fish” beaker = ______ C. Difference in number of drops of NaOH = _____ D. Amount of CO2 released by fish (from conversion Table 3-1) = _____ ml Why don’t you have to do any complicated thinking to know if this is a net increase or decrease? E. Amount of CO2 given off by fish PER HOUR = _____ ml/hr Lab 3-10 BIOLOGY 1001 NAME__________________________________ Lab day and time__________________________ Lab instructor_____________________________ LABORATORY 3: DATA SHEET 2 to be handed in at the beginning of next week’s lab USING THE SCIENTIFIC METHOD 1. Ask questions: Using your own natural scientific curiosity, while you are conducting the preliminary experiments with the Elodea and the goldfish, ask some questions about what is happening. Ask some questions about “What would happen if….” Example: what would happen if the goldfish were swimming in milk instead of water? 2. Develop hypothesis and make prediction: Using your questions above as a starting point, CHOOSE one experimental system (either the Elodea or the goldfish) and develop a hypothesis (or an educated guess) about what you think might happen if you did such and such. Make predictions based on this hypothesis. Example: the milk will have no effect on the aerobic respiration of the goldfish; the amount of carbon dioxide produced will be the same for a fish swimming in water as for a fish swimming in milk. Lab 3-11 3. Design experiment: Using a set-up similar to the one for your preliminary experiments, design an experiment to test your hypothesis. If you need additional materials, check with your lab instructor to see if they are available. If they are not, please develop a different hypothesis to test. Note that while the milk is a fine question, hypothesis, and prediction, you should be more careful in your hypothesis development and experimental design. You are free to kill the Elodea in the process of your experiment if you insist, but please treat the goldfish with more respect and honestly try not to kill or permanently harm it. In addition, keep your experiment SIMPLE. Do not try to test the effects of multiple changes because it can be impossible to determine which change had what effect (if the experiment was to compare warm water vs. cold milk, we couldn’t tell if the results were altered because of the temperature or because of the milk or because of both). DON’T FORGET TO USE APPROPRIATE CONTROL(S)! 4. Collect data: Design a table you can use to collect data from your experiment. Carry out your experiment and record your data in the table you designed. Draw your data table in the space below. Lab 3-12 5. Analyze data: Conduct any calculations necessary to analyze your data in a meaningful way that will permit you to determine if it supports or does not support your hypothesis. Don’t forget to convert to rates (for example, per hour) so that you can compare treatments. 6. Draw conclusions: Do your data (remember that data is plural and datum is singular) support or discredit your hypothesis? Please remember that you can NEVER “prove” that your hypothesis is correct. Lab 3-13 WRITING A LABORATORY REPORT By filling out Data sheet 2 on the different steps of the scientific method that you used, you have made it relatively easy for yourself to write a laboratory report based on your experiment. Your lab report does not need to be very long in length, but it does need to follow the instructions below very carefully. Laboratory reports follow the same general format as articles in scientific journals. In learning to write a lab report, you are learning a skill that will be valuable when you become a professional scientist. However, even if your life’s goal is not to become a scientist, the process of writing a lab report is useful AND FUN! (Well, maybe that’s stretching it a tiny bit, but give it a try….) The following instructions on writing a lab report are slightly modified from those at the Academic Writing Center at Hamilton College. The original version can be found at http://www.hamilton.edu/academics/resource/wc/bio_lab.html. A sample annotated lab report written using these instructions, can be found at http://www.hamilton.edu/academics/resource/wc/sample_bio.html. Lab Reports for Biology Format Please follow the instructions given below when writing lab reports for this course. Don't hesitate to ask if you have questions about form or content. Above all, remember to write with precision, clarity, and economy. Writing Your writing should be in full sentences and easily understood. It should conform to the conventions of standard written English (sentence form, grammar, spelling, etc.). Good writing is as important in science as it is in other disciplines because one's ideas have little impact, no matter how important they may be, if they are not well communicated. While style is mostly an individual characteristic, everyone should strive for presentations that are easily understandable as well as grammatically correct. One reason for emphasizing clarity is that writing and thinking are closely related; as many people have said, "fuzzy writing reflects fuzzy thinking." When people have difficulty translating their ideas into words, they generally do not know the material as well as they think they do. Style Lab 3-14 Scientific writing is usually in the past tense because one reports on experiments that have been completed. The writing should not be too self-referential (e.g., "I ground up the..."), although you may use the word "I" if doing so makes the writing easier to read. Writing that is predominantly in the passive voice is deadly to read (e.g., "acorns were eaten by the squirrels"), so use the active voice as much as possible (e.g., "squirrels ate the acorns"). Remember: past tense, active voice. Presentation The first page of a lab report should be a title page with the title of the report, your name, the date, the course (e.g., BC1001 Revolutionary Concepts Laboratory), your lab section (e.g., Monday 14pm), your lab instructor, and your lab partners. There should then follow text that is a minimum of two pages and a maximum of five double-spaced, typewritten pages in length (tables, figures, and references do not count in this total). All writing should be on only one side of the page, and the reports should be stapled in the upper left-hand corner. The best length is shorter than the maximum, so don't expand a shorter report to reach a five-page limit. It is important to write concisely. The report must be typed or word-processed. Neatness and clarity of presentation are almost as important as clarity of thought. Please do not use an interesting font or (especially!) font size to try to cheat on the length; nor should you use interesting margins. 12-point Times is a standard font most often used in academic settings. Audience Write the report as if you were writing to other students who are taking a similar course but have not done this experiment. Assume that they have some familiarity with the subject matter but no expertise. Do not write specifically for the instructor. Collaboration You may talk about the lab exercise as much as you like while in the laboratory. However, it is essential that you write your own report in your own words. You may and are encouraged to discuss the experiment itself with anyone at anytime to ensure that you have understood it. References If you use outside sources (including your textbook and your lab handout), and you should, then cite those sources in the body of the report and list the references in a literature cited section. Citations should be made with a standard scientific format (not by footnotes); cite the author and date of publication only, so that a quick look at the Literature Cited can provide the reader with all necessary information. When there are more than two authors, simply list the first author and et al., along with the date. You should not use direct quotations from the references; paraphrase information and give credit to the source of the idea. The following are sample citations: "Garrett (1989) showed that a gene in yeast ..." "... is found in the urinary bladder of the turtle (Gapp et al., 1990)." "... as reported recently (Miller, 1986; Pfitsch & Pearcy, 1989)." You should list a reference for every idea not your own. Plagiarism is more than copying Lab 3-15 material word for word; it is also using someone else's ideas or phraseology without giving reference to the other work or other person. Fortunately, the reference format is so simple that it is very easy to include references to all the work that one has used (Williams, 1983). If the idea is not published but is provided by a lab partner or someone else, give the reference as a personal communication (N. Cutler, pers. comm.). Be aware of the difficulties that arise when one uses material from another source and changes only a word here or there without acknowledging the source. Such actions are plagiarism, even though the statement may not be word-for-word the same as in the original. Just remember the basic rule: list a reference for every idea or statement that is not your own. Format There are four fundamental sections to a scientific report, with acknowledgments, literature cited, and appendices being additional sections. An underlined heading should be given at the beginning of each section (optional for the introduction). Keep in mind that the lab report is parallel to the experimental process (D. Flynn, 1988): Experimental Process What is the problem? How did I solve the problem? What did I find out? What does it mean? Lab Report Introduction Materials and Methods Results Discussion Title Your title should be DESCRIPTIVE, explaining what you did, how you did it, and what you found as much as possible (while it sounds contradictory, you should still be concise.) Bad title example: Milk and Goldfish. Good title example: Milk Decreases the Release of Carbon Dioxide from Goldfish, as Measured Indirectly Though the pH Indicator Phenolphthalein. Introduction Begin with broad statements, including enough background information (with reference to outside sources—most likely your textbook in this case) to set the stage for your experiment. Then narrow down to your particular study, explaining why it is of interest. Specify the objectives of the experiment, and make your hypotheses clear. One to three paragraphs is usually sufficient. Do not regurgitate the lab handout; write your own introduction. Materials and Methods (or just Methods) Summarize briefly the entire process that was followed and the materials that were used, and then refer to the lab directions and to any flow charts you have included for the details. Don’t forget to cite the lab handout! Do note any differences in the procedures you actually followed from what was specified in the lab directions. Anyone who reads your report should be able to duplicate the experiment. This section should be a small part of the report, so don't expand endlessly. Do not include results here. Results Lab 3-16 The data and results are given here in summary form. All results should be described in a narrative; don't just list measurements. One of the commonest mistakes beginning students make is to omit the narrative in the results section. The narrative should be more than just saying, "Table 2 shows the percentage of students with different blood types." You should state and explain the actual results, e.g., "Most students had type O blood, while the fewest had type AB (Table 2)." Data must be presented in figures (graphs) and in carefully planned tables, rather than as raw data All tables and figures should be titled and numbered sequentially, and the axes should be well labeled with clearly marked units. In addition to the title, each table and figure should have a legend (1 to 3 sentences) that explains what is being presented. A sample table and figure are given at the end of this handout. If the whole thing can be typed, it is a table; if lines have to be drawn, then it is a figure. Each table and figure should be put on a separate page and referred to by number in the results narrative. Tables and figures follow the text of the report (after the literature cited). Sample calculations may be included in an appendix at the end of the report. Discussion In this section the results should be interpreted and their significance explained. Begin the discussion by interpreting your specific results and end it more broadly by placing your results in context. Don't declare the experiment a success or failure; evaluate the results in view of the purpose of the experiment. If erroneous results were obtained, discuss the results you expected as well as those you received. You may also compare methods or discuss difficulties, but if you list sources of error, you should estimate how important each source of error may be. If you were to do the experiment again, what if anything, would you do differently? It is inappropriate to include statements such as "I learned a lot from this experiment..." The discussion is a very important section; it is your chance to show how well you understand the ideas and techniques involved and to relate your results to the ideas expressed in outside sources (the literature cited). Acknowledgments The acknowledgments section is optional. If you wish to thank someone, such as a lab partner or a tutor at the Writing Center, for help in understanding the experiment or in organizing the report, you do so here. Scientists regularly acknowledge others for helping with experiments or commenting on written drafts. Literature Cited List any publications referred to in your paper alphabetically by first author; do not number them. Every item in your bibliography should be referred to in the body of your paper, or it shouldn't be listed at all. If you use information from an intermediary source, you should list the original reference but should also note the intermediary: "...cited in...". We will use the following standard forms (some journals use variations of these), shown in order for (1) an article with one author, (2) an article with more than one author, (3) a book, and (4) a chapter from an edited volume: Reynolds, P.D. 1992. Mantle-mediated shell decollation increases posterior aperture size in Dentalium rectius Carpenter 1864 (Scaphopoda: Dentaliida). Veliger 35:26-35. Gapp, D.A., R.N. Taranto, E F. Walsh, P.J. Favorito, and Y. Zhang. 1990. Insulin cells are found in the main and accessory urinary bladders of the painted turtle, Chrysemys picta. J. Exp. Zool. Lab 3-17 254:332-337. Stokes, D., L. Stokes, and E. Williams. 1991. The Butterfly Book. Little, Brown and Co., Boston. 96 pp. Pearcy, R.W., and W.A. Pfitsch. 1994. Consequences of sunflecks for photosynthesis and growth of forest understory plants. Pages 343-359 in E.D. Schulze and M.M. Caldwell, editors. Ecophysiology of Photosynthesis. Springer Verlag, New York. Appendix Appendices are optional. You may use them to include your laboratory handout, sample calculations, sets of raw data, etc. Final Check The last thing to do before turning a report in is to read it. Correct all typographical errors and other mistakes and ensure that you have said what you want to say! These instructions on how to write a lab report were written by E.H. Williams, Hamilton College, with modifications by D. Gapp, N. Cutler, and E. Cuebas-Incle. [with further modifications by M. Olney, Barnard College.] Note that this is not the information you should cite for your experimental design—only what you should cite if you are saying where you got the instructions on how to write a lab report. Lab 3-18