ࡱ> @ <jbjbqq oj5gl\\\\\\\4....hd.O::::::::OOOOOOO, R *TO\:::::O\\::::\:\:OJT\\\\:ON 1D\\ K,T..xFL KOO=GTT KThe scientific method follows these steps: Observation or review of previous results. Pose questions. Develop a hypothesis. Identify variables and design experiment to answer the question or support a hypothesis. Perform experiments and gather results that support/reject the answer or hypothesis. Persuade your peers that your results are valid and of value. The first step to discovery is observation. This may be the observation of work, previously done by others, that gives you an idea that lets you discover more about an object or process, or it may be the initial observation of a new species that has never been described. Either way, a careful review of the current knowledge for the problem being investigated often leads to additional questions. Formulating good questions are the key to designing good experiments. Not all questions can be answered by the scientific process. Questions that address values are especially unsuitable because no objective experimentation can generate answers to them. For example, how do you design an experiment to answer a question like: Does Mary have a kind heart? How do you define "kind"? Now consider the following question: Does Mary have a four-chambered heart? What are the characteristics that make the second question scientifically answerable but not the first one? To properly deal with questions, the scientific process involves establishing an experimental design. For example, consider the question: How fast will bacteria grow? This is a very general question to which there are many answers because there are multiple factors that influence bacterial growth. You need to refine that question by defining the variables that influence growth, like: What kind of bacteria? What media will be used? How many bacteria should be used? How long will the bacteria be grown and how often should they be observed? What will be the temperature of the growth media? And so forth. A properly framed question may be: How many E.coli cells are present each hour after a colony is inoculated to a 1 liter flask containing 250 mLs of YT media shaken at 200 rpm in an incubator held at 37C? Notice that holding some variables constant, such as cell type, media type, inoculum quantity, and temperature allow the growth (dependent variable) to be observed over time (independent variable). The dependent variable is one that provides "measurements" that will address the answer you are seeking (it is the responding part of your experiment). For example, using the question above of "How many E.coli cells are present each hour after a colony is inoculated to a 1 liter flask containing 250 mLs of YT media shaken at 200 rpm in an incubator held at 37C", the number of bacteria is the dependent variable because that is the information you are seeking. The independent variable is time in this case because your question asks how bacteria are present each hour and you control how many hours the experiment will run. It is the manipulated part of your experiment. The experiment must go for more than one hour because you cannot obtain a growth rate for bacteria from doing an experiment only one hour, no more than weighing one person tells the average weight of humans. If you had asked, "how bacteria are present after 6 hours at several different temperatures", then temperature would be the independent variable instead of time because you would be in control of establishing the temperatures used. Normally, you deal with one independent variable at a time. You can properly frame the experiment by asking and also being able to propose an answer as to what effect the independent variable has on the dependent variable. Incidentally, you also cannot answer a question about a group of bacterial strains by using only one bacterial strains in your experiment. So, you must also ask: How many bacterial strains should be used to obtain a valid representation of the species population in question? Statistical procedures are usually involved in these decisions. The control variables are those influences that need to be held constant because variation in these influences would themselves change the outcome. In the above question "How many E.coli cells are present each hour after a colony is inoculated to a 1 liter flask containing 250 mLs of YT media shaken at 200 rpm in an incubator held at 37C", the control variables would be the amount of inoculum, the volume of media, the size of the flask, the shaker rmp and temperature. When developing an experimental design, ask yourself, "What are all the components or conditions that might influence the outcome of the experiment. Try to keep all of these components or conditions constant except for the independent variable. In designing experiments, you should consider the role of defining proper treatment ranges, including the proper controls and using an appropriate number of replicates or duplicates. TREATMENTS When data are produced from experimentation and reflected upon, one can make predictions about how variables affect one another. From the results of the above experiment, assuming that the design was credible, one should be able to predict what effect time has on the number of bacteria produced from a single colony. In another example, if a set of data was obtained about how many bacteria were present after growth for six hours at temperatures ranging from 15-40 C (what are the dependent/independent variables?), the data can be displayed as in Figure 1 and conclusions drawn.  Figure 1. Number of E.coli cell growth at 6 temperatures. The results shown graphically in Fig. 1 suggest that as temperatures increased the more the bacteria grew. You might now be able to predict (extrapolate from the figure) that the number of E.coli would reach 3 x 10^6 or more at 45 C or higher. However, if the experimental design originally had included 45 C, the results in Figure 2 may have been produced instead.  Figure 2. Number of E.coli cell growth at 7 temperatures. In the first instance, your prediction seemed to be reasonable within a certain temperature range (e.g. 15-40 C), but at higher temperatures perhaps the bacteria ceased to function from heat stress or simply died. You would not have known this had you not included 45 C in your design. In other words, you must establish appropriate levels of treatments for the independent variable. A pilot study and/or extensive reading may be required to determine if your experimental design is properly framed or has proper boundaries. CONTROLS Another crucial part of experimental design is to establish controls. What If you ask the following question: "Will a restriction enzyme cut this DNA"? Obviously, to address this question in your experiment, you are going to add a restriction enzyme to the DNA. However, how will you know if the restriction enzyme made any difference unless you also expose the DNA to the same conditions without the enzyme? The latter would constitute your controls. You can identify this part of your experiment if you will always ask: compared to what? For example, to conclude that "sugar water is sweet" requires that you also sample water without sugar to provide a known reference point from which to draw a conclusion (e. g. water with sugar compared to water without). Both elements must be included in the experimental design as you frame your question and experiment properly. REPLICATION Finally, one must be able to repeat (replicate) an experiment and produce the same results if a question asked is to be answered confidently. For example, to state that an apple will fall down to the ground from the tree (as opposed to up) is easy to predict because the Law of Gravity is so well proved. We have seen enough apples fall down to dispel any doubt in our minds about which direction apples fall. In other words, the experiments in which apples were dropped were replicated so many times that, in terms of probabilities, the conclusion that gravity works is no longer challenged. However, if one performs an experiment with one lizard and asks "how many crickets does the species eat per day"?, then how many lizards would one need to feed to conclude confidently that this species eats x number of crickets per day? Even then, one would have to say x plus or minus y because the number " x " will be an average or mean based on a set of numbers with a range of values (+/- y ). One might also wonder what other variables affect how many crickets lizards eat per day (think about it). To replicate is to redo the experiment; to duplicate is to have many identical experiments going on simultaneously or concurrently. For example, to feed one lizard daily for ten weeks constitutes a daily replication of a feeding regime; however, to duplicate would be to feed 5 lizards in 5 different cages daily for ten weeks. Duplication broadens your experimental database, a positive attribute of correct experimental designs. Sources of Variability What are some of the sources of variability that can have an influence on your data? Certainly, any uncontrolled variable can introduce a great amount of variability. But even when the all environmental variables are controlled, there are small errors in pipetting, weighing or even machine errors that can combine together to add variability to duplicated treatments. Practice leads to improvement in personal measuring techniques and frequent re-zeroing of machines can reduce machine induced errors. To further improve your accuracy, avoid distractions and fatigue when performing your experiments. Example Problem If we were faced with the problem that our computer didnt work, how might we approach the problem and resolve it? Lets start with the Experiment Template shown below and walk through how we might fill it out. I will put my running commentary in italics and the template headings in bold to distinguish them from the text that we fill in. Title: Date: Problem: The monitor on my computer does not light up when I turn the power strip on. Items at your disposal: Computer and monitor, power strip, circuit breaker box. Protocols Available: Switch settings Cord plugging Circuit breaker settings. Question: Is the monitor on/off switch indicator light on? Is the monitor cord plugged securely into both the power strip and the monitor? Is the power strip plugged in and does the on indicator light turn on when you flip the strip switch? Is the circuit breaker for the plug you are using tripped off? Is there power anywhere in the house? Other preliminary questions: Is the equipment defective? Assume that it is not. Hypothesis If the monitor on/off switch is off the monitor will not light up. Try toggling the on/off switch to see if the power indicator light comes on. If not, then there is not power to the monitor. If the monitor cord is not plugged securely into both ends then the monitor will not work. If both ends are securely plugged in and the monitor does not work then there is no power to the plug, check the powerstrip. If the power strip indicator light is not on then it is not plugged in or turned on and there is no power going to the monitor. Plug it in and turn on the power strip. If the indicator light is still not on then there may be no power to your plug. Check the plug circuit breaker. If the circuit breaker is tripped then there will be no power to the monitor. Reset the circuit breaker. If the monitor is still not on then check to see if there is electricity on anywhere in the house. If the power is out to the house then the monitor will not come on. Call your electric supply company and report the power outage. If there is power in the house, then the monitor or the circuit breaker may be defective. Try another plug. Variables: Independent: Monitor on/off switch is either on or off. Dependent: When on, Monitor will light up. Independent: Monitor cord in either plugged into monitor and power strip or not. Dependent: When plugged in the Monitor will light up. Independent: The power strip is plugged into the house circuit and indicator light is on or off. Dependent: When indicator light is on the Monitor will come on. Independent: The circuit breaker is either tripped or not tripped. Dependent: When the circuit breaker is not tripped the monitor will come on. Independent: The power is either on or off in the rest of the house. Dependent: When the power is on to the house the monitor will come on. Variables to control: None. Treatments: Monitor switch on/off. Monitor cord plugged or unplugged Power strip plugged or unplugged and on/off Circuit breaker tripped/ untripped House power on/off Treatment MS onMS offCord PluggedCord Un pluggedPSOnPS OffCB TripCB Not tripHP onHP offMonitor conditionMonitor SwitchXMonitor switchX(Off)Monitor cordXXMonitor cordXX(Off)Power stripXXXPower stripXXX(Off)Circuit breakerXXXX(Off)Circuit breakerXXXXHouse powerXXXXX(Should be on!)House powerXXXXX(Off)Controls: The computer monitor has come on before when the power strip switch is turned on. You could check the monitor by plugging it into another plug that is known to have power. Graph: No graphs needed. Calculations and Analysis: No calculations needed Results for each Hypothesis: Fill in Monitor condition in table above. Answer to the question: It depends on which hypothesis solves the problem. Review the consistency of your results with previous work or work of peers. Note the reasons for discrepancies. Compare results to the results of others seeking to solve the same problem. 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