Writing Assignment: write the Results and Discussion sections Due Friday of week 5 at 5pm (450 – 1000 words, incl citations) For detailed guidelines see the assignment rubric posted on Canvas! Title: concise but descriptive “Introduction” : State just your null and alternative hypotheses “Materials & methods”: state which statistical test you are using and which quarter/section your data comes from You are NOT writing these first two sections, just including this info Results section: • Describe the results of “your” experiment, including statistics • Do NOT draw interpretations or conclusions – just state your results! • Do NOT give raw data Discussion section: • Reject/fail to reject your null hypothesis • Discuss results and their implications – how does this relate to work that has already been done in this area? Cite at least one relevant study here. • Include thoughtful suggestions for further research Figures are optional but can be helpful for presenting your data! If you include figures/tables make sure they are referenced in the text. Submit on Canvas Lab Manual (R): Metabolism in Goldfish Objectives 1. Propose a testable hypothesis related to metabolic rates. 2. Design an appropriate experiment for testing your hypothesis and collect the relevant data, using goldfish. 3. Analyze and present your results and draw a conclusion concerning your hypothesis in a written scientific style research paper. Introduction All living things carry out metabolic processes, where metabolism is defined as the total chemical activity within that organism. The chemical activity which occurs is the sum of all anabolic and catabolic processes. Anabolic pathways synthesize important chemical building blocks, whereas catabolic pathways serve to break down molecules. The energy required to perform these reactions is obtained through respiration. Through respiration, organisms obtain oxygen, whose rate can then be measured to correlate with metabolic rate. Metabolic rate, however, can also be influenced by an organism’s body temperature. In lab this week, you will explore how different variables affect the oxygen consumption rate in goldfish. Goldfish are poikilothermic organisms whose body temperature changes when the environmental temperature changes, thus making the organism’s metabolic rate easier to manipulate. Each group will generate a hypothesis about your assigned environmental effect on the metabolic rates in goldfish. You will then design an appropriate experiment for testing the hypothesis. Finally, you will be given a set of data to analyze using a paired t-test. (This test is different from the unpaired test used in the Scientific Methodology and the MIT lab. See Appendix C for a detailed example.) This lab will be the basis of your second writing assignment. You will be given an environmental variable and its accompanying data in class. However, you are free to choose a different environmental variable when forming a hypothesis for your paper. You will use the metabolism database to test your hypothesis. You will analyze the results, as well as provide interpretations and conclusions for the experiment, in your paper. Please note: This is the unedited laboratory manual for the LS23L metabolism lab. You are being given the entire text in order to facilitate your writing process for Writing Assignment #2. Dr. Pfluegl will provide supplemental instructions on CCLE for how he would like you to complete this remote lab. Laboratory Exercise Experimental Materials Used in the LS23L Wet Lab ● ● ● ● ● ● ● ● ● ● ● Experimental variables Balance Thermometer Chamber with sensor Goldfish aquaria Nets Dish bins Beakers dH2O wash bottle Lamps Fish water carboy Procedure The Computer Program This lab exercise has an online tutorial to guide you as you formulate your hypotheses and predictions and learn about experimental strategies by which to test them. It is best to begin the lab by browsing through this tutorial. This will help you think of ideas for your writing assignment. The tutorial can be accessed from our lab website (https://ls23l.lscore.ucla.edu/TUTORIAL/?METABOLISM). The Metabolism Chambers You will be conducting your experiment(s) using goldfish in a small oxygen chamber. The oxygen chamber has a built-in probe that provides a continuously updated measure of the oxygen concentration in the chamber’s water. Because it is a closed system, the chamber will contain less and less oxygen as the fish utilize the oxygen that was initially in the water. Preparing the Metabolism Chambers for Experiments Keeping the Water Oxygenated for the Fish For each of your trials the oxygen chamber should contain a total of 400 milliliters fish water to ensure that the fish have enough oxygen. Because oxygen is depleted over the course of a trial, you must refresh the water between each trial. To do this, leave the fish in the chamber and gently pour off at least 100 milliliters of the oxygen-depleted water into your beaker. Then add the same volume of fresh fish water back into the chamber. If your experimental variable is dissolved in fish water, you can use fish water treated with your variable instead of plain fish water. Running LoggerLite From the computer desktop, open up the LoggerLite file called LoggerLite-metabolism expt. PLEASE NOTE: The dissolved oxygen probe needs to warm up for at least 10 minutes before you acquire data. Make sure that the probe is connected and then open up the LoggerLitemetabolism expt file BEFORE going to get your fish. Leave the program open to let the probe warm up. If you close the program, you will need to warm the probe up for another 10 minutes before resuming your experiment. To Run the Experiment: 1. Add the specimen and sufficient treated water to the oxygen chamber and secure the plunger lid. Use caution in trying to eliminate any bubbles inside the chamber. Give the fish two or three minutes to acclimate before beginning to collect data. 2. To begin recording data, click on the large green arrow button with the word “Collect.” You will see the quantity of dissolved oxygen change over time. 3. Use this data to create a graph in Excel or Google Sheets During your experiments, the oxygen probe will be assessing the oxygen concentration in mg/L. As more and more oxygen is used by the fish, the value will drop. The LoggerLite software will produce a mini-spreadsheet, continuously updated, that indicates the actual oxygen concentration in the water. For each trial you will need to graph these data points and add a trendline. The slope of the line will indicate the change in oxygen concentration over time for that trial. Figure 1. Example of LoggerLite data exported to Excel graphed and annotated. Designing an Experiment What Sorts of Questions Can You Ask? In the lab you will have access to materials that will allow you to alter the water temperature, the light intensity, and other variables. There are some experiments you can do using chemical manipulations. These manipulations have the potential to harm fish; therefore, the following guidelines for maximum dosages must be followed. Use the specially prepared fish water AT ALL TIMES. Ordinary tap water contains chlorine, which will kill fish. To save time and trouble, whenever you choose a variable that involves changing the chemical nature of the water for the fish, you should use half the treated water in the first trial and save half for use in the second trial. In doing so you will not need to spend time to prepare the test water again. Salinity: The maximum amount of salts that goldfish should be exposed to is 10 ppt (parts per thousand), which is the concentration of seawater your TA will provide. Temperature: The maximum level of temperature change allowed is ±10℃. You can choose either the colder water temperature or warmer water temperature as your experimental group. The control is room temperature fish water. Lighting: There are two variables that your group can choose, either with extra light or with black plastic covering the test chamber. The control is the normal ambient light. Caffeine: 50 mg of a caffeine tablet will be dissolved in fish water before being transferred into test chamber. Use half of the treated water for the first trial and half for the second. Nicotine: 1.5 g of tobacco is infused in fish water. Use half of the treated water for the first trial and half for the second. Remember that you are not restricted to these experiments and are encouraged to use your imagination (while avoiding cruelty and being intrusive to the goldfish). What Will A Good Experiment Entail? It is critical that you propose a hypothesis that you can answer definitively. Do not rush into data collection before carefully considering how testable your hypothesis is (especially given the time constraints you are working under in the lab). Control Group ● Should the fish be the same size or different sizes? ● Should the control fish be the same fish as in the experimental group? ● Should the control or experimental measurements be made first? Experimental Group ● What sort of manipulation will you be making? ● Can you imagine all possible results? Do you know how you will interpret them? ● Are you unintentionally altering more than one variable at a time? ● Is your experiment interesting? For example, we all know that putting a stir bar into the chamber and turning it up to a high speed would cause your fish to increase their metabolic rates. Try to imagine an experiment with two features: (1) you don’t know what the results will be, and (2) you (and your TA) would like to know what the results will be. Figure 2. Pictorial representation of a good experimental set-up. Note that while two trials are performed, the same two fish are used for the control group and experimental group in each trial. Figure 3. Pictorial representation of the student groups. Each classroom “bench” (shown here in blue), will have four groups who decide on and use the same experimental condition during their experiment (i.e. Condition 1 is darkness and Condition 2 is salinity). How Can You Anticipate Potential Problems? Create a flowchart describing the order in which you will perform the various parts of your experiment, from hypothesis to experimental setup and execution, to data analysis, and the components of each of these parts. A good flowchart will help you anticipate sources of error and, thus, organize your experiment to minimize problems. (See Figure 9 for an example flowchart.) Consider the following as you develop your own flowchart: a. Try to envision how you will analyze your data before beginning the experiment. ● What comparisons will you make? ● What values will you graph? ● How large a sample size do you need? That is, how many trials do you need to run in order to know that the effect you observe is repeatable, and, therefore, likely real? This is particularly important if you intend to test differences between control and experimental treatments statistically. If so, have you planned enough trials to adequately estimate means and standard deviations? ● What graphs do you want in your research paper? b. Try to imagine any criticisms that a reader of your paper might have and address them. ● Have you quantified oxygen use in a way that makes sense? Which makes more sense for your experiment: rate of oxygen usage (i.e., the slope of the line plotting oxygen concentration vs. time), absolute change in oxygen use over some fixed period of time, or oxygen consumption per fish (or per gram of fish) per unit time? ● Have you used the proper control group, given the way you chose to quantify oxygen use? ● Have you controlled all extraneous variables such that the treatment and control conditions differ only with respect to the variable under investigation? ● Does the amount of water in the chamber affect your measurements of oxygen consumption? Analyzing and Presenting Your Data What Sort of Data Output Do You Get from the LoggerLite Program? You will get a table of data with two columns. The first column is the time in seconds and the second column is the oxygen concentration in the water during that run. After each run, you can import the data into Excel to produce a linear regression equation and chart. Remember to record all data after each run. How Can You Present This Data in an Interesting Way? Alternative 1 Use the raw data. For example, a group of students attempted to test the null hypothesis that oxygen consumption rates of male and female fish do not differ. Two groups of fish were enclosed in the chamber separately; one consisted of three female fish and the other of three male fish. Oxygen consumption rates of each group of fish were measured only once. That is, only a single trial per group of fish was performed. The raw data for this experiment included oxygen concentrations through time for each group. The students entered their raw data (the time and oxygen concentration from each trial) into a spreadsheet program (such as Excel), made a scatter plot, and fit a best-fit trendline like the one seen in Figure 4. Is this experiment well controlled? Can an observed difference in oxygen consumption rates between males and females be attributed unquestionably to differences in sex? What other factors might account for an apparent difference in oxygen consumption rates in this experiment? For example, were the males and females exactly the same size in both groups? What effect might size have on oxygen consumption rates? Can these effects be distinguished from the hypothesized effects of sex in this case? Figure 4. Metabolic rates of male and female goldfish measured as oxygen consumption. Each line reflects the collective metabolic rates of three fish (n = 1 for each treatment). Alternative 2 The students performed a simple analysis of the data by calculating the rate of oxygen consumption as oxygen lost per hour [(O2 time begin – O2 time end)/total time]. Data such as this can be presented using a column, bar, or scatter graph. See Figure 5. Figure 5. Metabolic rates of male and female goldfish measured as oxygen consumption and presented as average rate of oxygen consumption per hour. Each bar represents the collective metabolic rates of three fish (n = 1 for each treatment). Alternative 3 The students used the slopes of the linear regression lines calculated by the LoggerLite program (Figure 6). In their lab report, the students discussed what these differences in slopes meant, e.g., what did a large negative slope mean biologically? How were the slopes calculated? If you use LoggerLite to fit a curve to all data collected, the absolute value of these slopes should be the same as those values calculated in Alternative 2. Why? Hint: How do you calculate the slope of a line? However, if you use LoggerLite to fit a curve to only a portion of the data, the values may differ. Why? Figure 6. Metabolic rates of male and female goldfish measured as oxygen consumption and presented as the slope (mgO2 per liter per hour). Each bar represents the collective metabolic rates of three fish (n = 1 for each treatment). Alternative 4 Students performed multiple trials within each of the two treatments (female and male). This enabled them to calculate the average rate of oxygen loss across trials and then compare these averages between treatments. To do this, they measured the oxygen consumption rates of four different groups of female fish and four different groups of male fish; each group consisted of three fish. Each different group of three fish constitutes a “trial.” This produced an n = 4, i.e., a sample size of four trials per treatment. (A “treatment” is the condition of being male or female.) They then calculated the rate of oxygen lost per hour (as in Alternative 2) for each trial individually and averaged these values for each treatment. The results are presented in Figure 7. What are the advantages of repeated trials over single runs (as in Alternatives 1, 2, and 3) with respect to the confidence you have in your results? The error bars presented in Figure 7 are standard deviations, and, as such, reflect the variability in metabolic rates among trials within a treatment-that is, how much oxygen consumption rates differed between different groups of fish within a treatment. Figure 7. Metabolic rates of male and female goldfish measured as oxygen consumption and presented as average rate of oxygen consumption per hour (± standard deviation, n = 4 for each treatment). Alternative 5 The students controlled for differences in the size (weight) of fish among trials and between treatments by calculating the cate of oxygen loss per kilogram of fish. To do this, they divided the rate of oxygen loss (as calculated in Alternatives 2 and 4) by the total weight of the fish in each trial (four trials per treatment as described in Alternative 4). They then averaged these weight-standardized oxygen consumption rates among trials within each treatment as in Alternative 4. Data such as these can be presented as a column, bar, or scatter graph. The students’ results are presented in Figure 8. Compare the results presented in Figure 8 to those in previous figures. Did standardizing for differences in fish weight alter the interpretation of the results of this experiment? Why or why not? Figure 9. Example flowchart of experimental design and execution. Cleanup ● ● ● ● ● Return fish ONLY (no used fish water) to the original aquarium. Follow the instructions for the chamber posted at each station. Make sure the chamber has enough dH2O in it to submerge the probe. Return all items to their original places and clean up your station. Log off your computer. The Scientific Style Research Paper for Metabolism (Refer to Appendix A for an example.) For this assignment, you will learn to write the second half of a scientific research paper. Because we cannot run the actual lab this quarter, you will be writing your text based on data chosen from the metabolism database, which contains all LS23L data from past quarters. You will form a hypothesis and then look at the metabolism database to find a lab section that performed the experiment with your environmental variable. Each lab section has two benches that performed two different experiments (Figure 3). For example, if you look at section 1A from Fall 2019, one bench chose cold water and one bench chose nicotine to run their experiment with. You can choose any variable that is of interest to you. If you would like, you may also write about data for your variable from a broader time period (i.e., the entire quarter in which your chosen section was held), but this must be IN ADDITION TO the data for your chosen section, so in this case you would need to present two p-values (one for your bench data, one for the broader time period). All data are available on our website and the link is provided in the “Metabolism in Goldfish” section on CCLE. For specific guidelines and deadlines, please refer to the Writing Assignment #2 rubric and Dr. Pfluegl’s presentation, both posted on the LS23L CCLE site. TITLE Short, concise, and relevant. INTRODUCTION You will not be writing an introduction section for this assignment. Instead, simply state your hypotheses (null and alternative). MATERIALS AND METHODS You will not be writing a materials and methods section for this assignment. Instead, simply state what statistical test was used to analyze your data. RESULTS Describe the results of the experiment, including your statistical results. You should never present raw data (individual measurements or the results of individual trials, for example) in a scientific paper. When deciding what data to include, think about what you would find useful if you were reading someone else’s study. Keep in mi…
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