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Biology& 100 Laboratory Manual For Mr. B’s sections Green River Community College Biology& 100 Laboratory Manual Mr. Brumbaugh 1 Revised Summer 2016 Preface This manual has been compiled and written for your enjoyment and learning as you work through the Biology& 100 course. The exercises selected by your instructor are meant to accentuate your learning of the basic concepts, ideas, and hypotheses concerning biology. Each exercise should reinforce the learning that is taking place within the confines of the lecture portion of the course. The exercises are designed to give you a “hands on” feel for the material presented in lecture. Please take to heart the following suggestions for your successful completion of Biology& 100. Spend time before lab reading each assigned laboratory thoroughly. This will allow you to organize your time during lab and to foresee pitfalls and pratfalls that could prevent you from completing the lab within the prescribed timeframe. As you prepare for the lab, jot down questions about concepts or procedures that you would want answered to facilitate your lab experience. Most labs will have pre-lab questions’, answer them to the best of your ability by reading each lab completely prior to lab to assist your understanding. Come prepared to discuss or turn-in the pre-lab questions. If you are assigned to an activity research group, plan a meeting time each week prior to the day of the lab to finalize the understanding of the procedures for the lab. Before leaving lab ensure that you have a good understanding of the principles so that you can finish the write-up or answer the questions associated with that lab. Approach lab with an open mind for learning and attempt to see the application of the information and the relatedness to the lecture material. Have fun and use the time to maximize your learning of the concepts of biology. Biology& 100 Laboratory Manual Mr. Brumbaugh 2 Revised Summer 2016 Biology& 100 Laboratory Manual Green River Community College Table of Contents and Appendices Please Take Note!! It is your responsibility to read each assigned lab prior to coming to the lab and to answer any pre-laboratory questions associated with the individual labs. Laboratories Safety and Emergency Procedures Laboratory 1 Principles of the Scientific Method Laboratory 2 Microscopic Observation of Cells Laboratory 3 Transporting Across Boundaries Laboratory 4 Pea Lab: Scientific Method Applied Laboratory 5 Energy Harvest – Fermentation in Yeast Laboratory 6 Mitosis and Online Karyotyping Laboratory 7 Mendelian Genetics Laboratory 8 Modeling DNA Structure, Replication, & Protein Synthesis Laboratory 9 Critical Thinking and Classification Laboratory 10 Paper Project pg. 05 pg. 07 - 22 pg. 23 - 38 pg. 39 - 51 pg. 53 - 59 pg. 61 - 71 pg. 73 - 79 pg. 81 - 93 pg. 95 - 108 pg. 109 - 117 pg. 119 – 123 Appendices Appendix Appendix Appendix Appendix Appendix Appendix Appendix A B C D E F G Biology& 100 Laboratory Manual Mr. Brumbaugh Criteria for Graphing Scientific Data Metric Conversions General Format for Writing a Scientific Paper Drawing: The Making of a Plate or Drawing How to Make an Oral Presentation How to search the Literature Animal & Plant Kingdom Highlights 3 pg. 124 - 125 pg. 126 pg. 127 - 131 pg. 132 - 134 pg. 135 - 136 pg. 137 - 139 pg. 140 - 145 Revised Summer 2016 First blank page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 4 Revised Summer 2016 Biology Laboratory: Safety Procedures and Emergencies 1. No open food or drink is permitted in laboratory rooms at any time, whether a lab is in progress or not. No eating, drinking, chewing of gum or tobacco is permitted. Never taste anything at all while in the laboratory rooms, unless it is a part of the lab activity (such as PTC paper). 2. Know the locations of the eye wash and shower stations, fire alarm, fire extinguisher, first aid kit, and emergency exits. 3. If you have any allergies (including latex and bee stings), please inform your instructor so that we can be aware of your needs during lab activities and field trips. 4. Safety instructions are given at the beginning of each lab activity. Always arrive on time so that you know what you are supposed to do and are informed of any specific safety concerns or safety equipment associated with the day’s lab activity. 5. Wear any required personal protective equipment and appropriate attire for lab activities and field trips (lab coat, apron, goggles, rain gear, No open toed shoes, etc.). If safety goggles are required for a lab that means they are required for the entire lab. 6. Stash book bags safely in the spaces provided in the front of the room so that they won’t trip people. 7. Report all illnesses, injuries, breakages, or spills to your laboratory instructor immediately. 8. Clean broken glass (glass that is not contaminated with any chemical reagents, blood, or bacteria) can be swept up using the dust pan and placed in the broken glass container. If the glass is contaminated in any way, keep the area clear to prevent tripping or laceration hazards, and consult your instructor for proper disposal guidance. 9. Notify your instructor if any of the equipment is faulty. 10. Clean up your entire work area before leaving. Put away all equipment and supplies in their original places and dispose of reagents and infectious materials in the designated receptacles. Disinfect your work surface if the lab activity involved any infectious materials. 11. Use the appropriate waste containers provided for any infectious or hazardous materials used in lab. 12. Safety information reagents used in the lab activities can be found in the Safety Data Sheets (SDS’s), which are available in a binder in the lab prep room. Know the location of the SDS binder. We (faculty and students) should be fully aware of the properties of the reagents we are using. Please use the SDS’s. If you cannot find the SDS for the reagent you are using in lab, inform your instructor. They are also relatively easy to find online. A Google keyword example is “Sodium Chloride SDS.” 13. Use caution with the lab chairs. Because they are on casters, they can roll away from you when you are standing at your workstation. Make sure your chair is where you expect it to be before sitting down. Do not use your chair as a means of moving from one part of the lab to the other. 14. Wash your hands before and after concluding the day’s lab activities. 15. I have read the above statements and will adhere to them while in lab. Signature: . Date: Print: Biology& 100 Laboratory Manual Mr. Brumbaugh . . 5 Revised Summer 2016 Biological Doodle Page Biology& 100 Laboratory Manual Mr. Brumbaugh 6 Revised Summer 2016 Laboratory 1: Principles of the Scientific Method Pre-lab Assignment Before coming to lab carefully read the following pages on the Scientific Method and Appendix A then answer the pre-lab questions, pages 21 & 22. Be prepared to hand in your responses to the prelab questions at the start of lab. Perspectives Biology is a dynamic field of study whose aim is to unravel the mysteries of life itself. Throughout history, humans have been curious about the world around them. Through the millennia people have observed the natural world and have asked, “Why?” Those that have advanced our biological knowledge the most, whether the great scientists of the centuries before us, such as Robert Hooke (discovered cells in 1665) and Charles Darwin (co-developer of the theory of evolution by natural selection in 1859), or modern molecular biologists such as James Watson and Francis Crick (discovered the structure of DNA in 1953), have certain traits in common. They had inquiring minds, great powers of observation, and they used a systematic approach first developed by Roger Bacon an English monk in 1265 to answer the questions that intrigued them, the scientific method, which is similar to how you look at the world. In this course you will have ample opportunity to develop your scientific skills. The weekly laboratory exercises are designed not only to stimulate your curiosity and heighten your powers of observation, but also to introduce you to and allow you to practice the scientific method. This laboratory activity will allow you to practice the scientific method as you study the factors that influence your heart rate and level of physical fitness. Let’s first learn a bit about the scientific method in more detail. Scientific Method The scientific method is neither complicated nor intimidating, nor is it unique to science. It is a powerful tool of logic that can be employed any time a problem or question about the world around us arises. In fact, we all use the principles of the scientific method daily to solve problems that pop up, but we do it so quickly and automatically that we are not conscious of the methodology. In brief, the scientific method consists of Observing natural phenomena Asking a question based on one’s observations Constructing a hypothesis to answer the question Testing the hypothesis with experiments or pertinent observations Drawing conclusions about the hypothesis based on the data resulting from the experiments or pertinent observation Publishing results (hopefully in a scientific journal!) Observations The scientific method begins with careful observation. An investigator may make observations from nature or from the written work of other investigators, which are published in books or research articles in scientific journals, available in the storehouses of human knowledge, libraries. Let’s use the following example as we progress through the steps of the scientific method. Suppose that over the last couple of years you have been observing the beautiful fall colors of the leaves on the vine maples that grow in your yard, on campus, and in the forests in the Cascade Mountains. You note that their leaves turn from green to yellow to orange to red as the weather turns progressively colder and the days get shorter and shorter. However, the leaves do not always go through their color changes on exactly the same days each year. Biology& 100 Laboratory Manual Mr. Brumbaugh 7 Revised Summer 2016 Questions It is essential that the question asked is a scientific question. I.e. The question must be testable, definable, measurable and controllable to produce verifiable data. For example, one would have a tough time trying to test the following question; “Did a supernatural force such as God create all life on earth?” Moreover, since the concept of God has many different meanings and definitions, it is difficult to define what or who God is from a science standpoint. Since this question is not a scientific question, and hence not testable, the courts of the United States have ruled that “creation science” should not be taught in science classes as has been demanded by various groups in this country. However, that’s not to say that God did not create life, it’s just not testable from a science viewpoint, but rather, a matter of faith. Now, back to the vine maple example...Being a curious and inquisitive person you ask, “What’s causing or stimulating the vine maple’s leaves to change color?” Hypotheses The next step in the scientific method is to make a hypothesis, a tentative answer to the question that you have asked. A hypothesis is an educated guess that is based on your previous observations. It’s a trial solution to your question that you will test through experimentation. Hypotheses are often stated in an “If... then...” statement. Now back to the vine maples. You have noted that vine maples change color in the fall on approximately the same dates each year, but this varies by a week or two each year. You hypothesize, since air temperature is not constant each year in the fall, the progressively cooler days in fall are responsible for stimulating the color changes. Therefore, you develop and wish to test the following hypothesis: If progressively cooler temperatures are responsible for stimulating the color changes in the leaves of vine maples, then vine maples placed in an artificially cooled growth-house (green house) should go through the same color changes as would the vine maples in nature, even if the length of day/night are held constant via artificial lighting. Testing Hypotheses via Experiments or by Pertinent Observations The next step of the scientific method is to design an experiment or make pertinent observations to test the hypothesis. In any experiment there are three kinds of variables. Independent variable: The independent variable is the single condition (variable) that is manipulated to see what impact it has on a dependent variable (measured factor). The independent variable is the factor that causes the dependent variable to change. E.g. the temperature the trees are exposed to is the independent variable in this vine maple example. The independent variable is the factor (i.e. experimental condition) you manipulate and test in an experiment. A great challenge when designing an experiment is to be certain that only one independent variable is responsible for the outcome of an experiment. As we shall see, there are often many factors (known as control variables) that can influence the outcome of an investigation. We attempt, but not always successfully, to keep all of the controlled variables constant and change only one factor, the independent variable or control treatment, when conducting an experiment. Once the parameters or limits (such as the amount, intensity, volume, or etc.) of the independent variable have been delineated then the independent variable becomes a controlled treatment for the experiment. Dependent Variable: The thing measured, counted, or observed in an experiment. E.g. the color of leaves is the dependent variable in the vine maple example. Control Variables: These are the variables that are kept constant during an experiment. It is assumed that the selected independent variable is the only factor affecting the dependent variable. This can only be true if all other variables are controlled (i.e. held constant). In the vine maple example: species of vine maple, age and health of the trees used, length of day, environmental conditions such as humidity, watering regime, fertilizer, etc. It is quite common for Biology& 100 Laboratory Manual Mr. Brumbaugh 8 Revised Summer 2016 different researchers, or for that matter, the same researcher, to get different and conflicting results while conducting what they think is the very same experiment. Why? In an experiment of classical design, the individuals under study are divided into two groups: an experimental group that is exposed to the independent variable (e.g. the group of trees that are exposed to the varying temperatures), and a control group that is not. The control group would be exposed to the identical conditions as the experimental group, but the control group would not be exposed to the independent variable (e.g. the control group of vine maples would be kept at a constant temperature, everything else would remain identical.) Sometimes the best test of a hypothesis is not an actual experiment, but pertinent observations. One of the most important principles of biology, Darwin’s theory of natural selection, was developed and supported by his extensive observations of the natural world. Since Darwin’s publication of his theory, a multitude of experiments and repeated observation of the natural world continue to support Darwin’s theory. An important hypothesis may become a theory after it stands up consistently to repeat testing by other researchers. A scientific theory is a hypothesis that has yet to be falsified and has stood the test of time. Hypotheses and theories can only be supported, but cannot be proved true by experimentation and careful observation. It is impossible to prove a hypothesis or theory to be true since it takes an infinite number of experiments to do this, but it only takes one experiment to disprove a hypothesis or a theory. Scientific knowledge is dynamic, forever changing and evolving as more and more is learned and new techniques are developed. Conclusion Making conclusions is the next step in the scientific method. You use the results and/or pertinent observations to test your hypothesis. However, you can never completely accept or reject a hypothesis. All that one can do is state the probability that one is correct or incorrect. Scientists use the branch of mathematics called statistics to quantify this probability. Publication in a Scientific Journal Finally, if the fruits of your scientific labor were thought to be of interest and of value to your peers in the scientific community, then your work would be submitted as an article for publication in a scientific journal. The goal of the scientific community is to be both cooperative as well as competitive. Research articles both share knowledge and provide enough information so that the results of experiments or pertinent observations described by those articles may be repeated and tested by others (verifiable data). It is just as important to expose the mistakes of others, as it is to praise their knowledge. Heart Rate and Fitness Exercise Goals of Lab Exercise Learn proper graphing technique To learn and apply the steps of the scientific method to answer questions concerning physical fitness Use a computer and heart rate monitor to measure the human heart rate Determine the effect of body position on heart rates Determine the effect of exercise on heart rates Correlate the fitness level of individuals with factors such as smoking, the amount of daily exercise, or other factors identified by students. Biology& 100 Laboratory Manual Mr. Brumbaugh 9 Revised Summer 2016 Introduction The Circulatory System The circulatory system is responsible for the internal transport of many vital substances in humans, including oxygen, carbon dioxide, and nutrients. The components of the circulatory system include the heart, blood vessels, and blood. Heartbeats result from electrical stimulation of the heart by the pacemaker (sino atrial node or SA Node), located in the heart’s inner wall of the right atrium. Although the electrical activity of the pacemaker originates from within the heart, nerves outside of the heart influence the rhythmic sequence of impulses produced by the pacemaker. Many things might affect the rate of the heart’s beating, including the physical fitness of the individual, the presence of drugs such as caffeine or nicotine in the blood, or the age of the person. The increase in heartbeat rate during exercise can be measured by monitoring the individual’s heart rate. As a rule, the maximum heart rate of all individuals of the same age and sex is about the same, yet the time it takes individuals to reach that maximum level while exercising varies greatly. Since physically fit people can deliver a greater volume of blood in a single cardiac cycle than unfit individuals, they usually can sustain a greater work level before reaching the maximum heart rate. Physically fit people not only have less of an increase in their heart rate during exercise, but their heart rate recovers to the resting rate more rapidly than unfit people. In this experiment, you will evaluate your physical fitness. An arbitrary rating system will be used to “score” fitness during a variety of situations. Tests will be made while in a resting position, in a prone position, as well as during and after physical exercise. Let’s now take a look at the Scientific Method. Materials Computer Stepping platform, 9” high Caution! Go-Link Interface Hand held Heart Rate Monitor Heart Rate Monitor Program Recorded Metronome Do not attempt this exercise if physical exertion will aggravate a health problem. Inform your instructor of any concerns that you may have and if you experience any issues following the exercise. Procedure Developing a Question, Hypothesis, and Experimental Procedure 1. In teams of 4, take a few minutes to discuss several specific questions about an independent variable related to cardiovascular fitness peculiar to your group. For example, you might ask: “Is there a difference in cardiovascular fitness between males and females? 2. Cardiovascular fitness will be assessed by determining and comparing the heart rate while standing, reclined, going from a reclined to a standing position, and before and after physical activity as outlined in steps 1 - 18, under Collecting Data from Test Subjects (page 11). 3. Write your group’s best question and hypothesis on the Report Sheet (page 15) and contribute your group’s hypothesis on the front board. 4. Determine which hypothesis that the entire class will attempt to answer. Record this hypothesis on the Report Sheet. 5. Design an experiment that will test this hypothesis. All teams will perform the same experiment. List the generalized steps (at least 5) of the experimental design on the Report Sheet. 6. Each group should recruit one subject for treatment 1 and another subject for treatment 2. One test subject will complete steps 1 – 18 under the direction of the investigators (other Biology& 100 Laboratory Manual Mr. Brumbaugh 10 Revised Summer 2016 group members) and then the other subject will be tested. One investigator (one of your partners) should record your data on the Report Sheet. The Set Up 1. Prepare the computer for data collection by opening the Biology with Computers software as follows: Plug the hand held heart rate sensors into the Go-Link connector then go to Start click on Programs open Vernier open Logger Pro go to File open Biology with Vernier open “Exp. 27” Heart Rate & Fitness. 2. Have your first test subject grasp the sensors while sitting, ensure that the direction of the arrow on the sensor matches the direction of the arrow on the receiver, and they are within 3 to 4 ft. of each other. Click the COLLECT button and continue collecting until the test subjects’ heart rate is steady and within the normal range for the individual—between 55 and 85 bpm. 3. Click on the STOP button to stop data collection when you have determined that all equipment is functioning properly. 4. To obtain the average heart rate, maximum heart rate, etc. during the test: Select “Analyze” on the menu bar at the top of the screen and click on “Statistics”. This will place a table onto the graph, find the mean in the box, and record on the appropriate data table on the Report sheet when actually collecting data. 5. Erase the data from this run and begin the experiment with your first subject. Collecting Data from Test Subject 1. 2. 3. Start with either subject (treatments 1 or 2) and follow steps 2 - 18. After step 18, each group should repeat steps 2-18 with a subject representing the other treatment category. Instruct the test subject to stand upright, click the COLLECT button, and begin taking data with the Heart Rate Monitor program. Wait until the heart rate becomes stable, record for I minute, and then record the subject’s heart rate in the proper column in Table 6A or 6B (page 16 or 17, respectfully) based on your assigned group number. Compare the subject’s standing heart rate to the values in Table 1. Assign fitness points based on Table 1 and record the fitness points on the Report Sheet. Beats per minute Fitness points Beats per minute Fitness Points < 60 - 70 12 101 - 110 8 71 -80 11 111 - 120 7 81 - 90 10 121 -130 6 91 -100 9 131 -140 4 Table 1 Fitness Points for Standing Heart Rate Use this table to assign fitness points based on the subject’s standing heart rate. 4. 5. Instruct the subject to recline on a clean table with their feet on the table and knees bent. Wait until the heart rate becomes stable, record for 1 minute, and then record the subject’s average heart rate on the Report Sheet. The subject remains reclined until step 6. Compare the subject’s average reclining heart rate to the values in Table 2, assign fitness points based on Table 2, and record the fitness points on the Report Sheet. Beats per minute Fitness points Beats per minute Fitness Points < 50 - 60 12 81 - 90 8 61 - 70 11 91 - 100 6 71 - 80 10 101 -110 4 Table 2 Fitness Points for Reclining Heart Rate Use this table to assign fitness points based on the subject’s reclining heart rate. Biology& 100 Laboratory Manual Mr. Brumbaugh 11 Revised Summer 2016 6. 7. 8. 9. Instruct the test subject to quickly stand up next to the lab table and remain still. Measure the subject’s peak heart rate upon standing (This takes a few moments and the number will appear in the heart rate box on the screen.) and then record it on the Report Sheet. Find how much the heart rate increased after standing by subtracting the reclining rate value in Step 4 from the peak standing value in step 6. Assign fitness points corresponding to your reclining to standing heart rate in Table 3 and record the fitness points on the Report Sheet. Stop data collection. Have the test subject stand and begin collecting heart rate data. Wait until the heart rate becomes stable (Initial standing heart rate), and then record the subject’s heart rate on the Lab Report Sheet. Heart Rate Increase after Standing Ave. Reclining rate (beats/min) 0–10 11–17 18–24 25–33 34+ 50–60 12 11 10 8 6 61–70 12 10 8 6 4 71–80 11 9 6 4 2 81–90 10 8 4 2 0 91–100 8 6 2 0 0 101–110 6 4 0 0 0 Table 3 Fitness Points for Reclining to Standing Use this table to assign fitness points based on the subject’s reclining to standing heart rate changes. 10. While holding the hand held sensor devices have the test subject step up and down on a low platform about 8 to 10 inches from the ground as follows: Place the right foot on the top step of the stool. Place the left foot completely on the top step of the stool next to the right foot. Place the right foot back on the floor. Place the left foot back on the floor. Repeat the above stepping cycle for 3 minutes 11. Use the recording of a metronome set at 96 beats per minute to ensure that all subjects maintain a constant step rate. The test subject should make one-foot movement for each beat of the metronome. 12. Record on the Report Sheet the subject’s average heart rate after 3 minutes of exercise. When the subject has completed the step exercise, quickly move to Step 13. 13. With a stopwatch or clock, begin timing to determine the test subject’s recovery time. During the recovery period, the test subject should remain standing and still. Monitor the heart rate and stop timing when the rate returns to the Initial standing heart rate value before the start of the step test (recorded in Step 6). Record the recovery time on the Report Sheet. 14. Assign fitness points based on the information in Table 4 (page 13). Record the fitness point value on the Report Sheet. 15. To calculate the endurance heart rate, subtract the initial standing heart rate before exercise (Step 6) from the average heart rate during exercise (Step 12). Record this heart rate increase in the endurance row on the Report Sheet. 16. Assign fitness points based on Table 5 (page 13) and record the value on the Report Sheet. 17. Total the fitness points for the subject and use Figure 1 (page 13) to determine an arbitrary fitness level and record on the Report Sheet. 18. Repeat steps 2 - 17 with a new test subject for the other treatment category. 19. As a group answer the questions on the Report Sheet (pages 15 through 20) and turn in one packet per group next week at the start of the lab. Biology& 100 Laboratory Manual Mr. Brumbaugh 12 Revised Summer 2016 Recovery Time (seconds) Fitness Points 0–30 14 31–60 12 61–90 10 91–120 8 121-150 6 > 150 4 Heart rate stabilized at a higher rate than the average standing value 6 before starting the step test Heart rate did not fall to within 6 to 10 beats/min. of the initial rate within 4 150 seconds after the cessation exercise Table 4 Fitness Points for Recovery Time Fitness points based on the subject’s total heart rate recovery time. Standing rate (beats/min) Heart rate increase after exercise 0–10 11–20 21–30 31–40 41+ 60–70 12 12 10 8 6 71–80 12 10 8 6 4 81–90 12 10 7 4 2 91–100 10 8 6 2 0 101–110 8 6 4 1 0 111–120 8 4 2 1 0 121–130 6 2 1 0 0 131+ 5 1 0 0 0 Table 5 Fitness Points for Endurance Fitness points based on the subject’s endurance rate. Fitness Scale Low Fitness 20 Very Fit Fit 30 40 50 60 Figure 1 Fitness Scale Use this scale is to chart the arbitrary fitness level for each subject. Biology& 100 Laboratory Manual Mr. Brumbaugh 13 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 14 Revised Summer 2016 Report Sheet Lab Section: Principles of the Scientific Method Exercise Group Names: . . . . . Question, Hypothesis, and Experiment: From Developing a Question, Hypothesis, and Experimental Procedure: Group Work: 1. Your group’s best question: Your group’s hypothesis: Class Work: 2. Hypothesis proposed by the class: Summary of the experimental procedure designed by the class: 3. List below the various components of the experiment designed by the class Dependent variable(s): Independent variable(s): Controlled variable(s): Control treatment: Biology& 100 Laboratory Manual Mr. Brumbaugh 15 Revised Summer 2016 Data: Treatment 1: Situation: Group Number Standing heart rate (beats/min) Reclining heart rate (beats/min) Peak heart rate upon standing (beats/min) Initial standing heart rate just before step test (beats/min) Heart Rate Average after step test (beats/min) Recovery time (seconds) Endurance (beats/min) Heart Rate 1 2 3 4 5 6 Fitness Points 1 2 3 4 5 6 Total fitness points Average total fitness points for treatment 1 Arbitrary Fitness Level Table 6A Treatment #1 Data Table Record your heart rate and fitness points on this table captured from Treatment #1 subject. Miscellaneous Notes and Observations: Biology& 100 Laboratory Manual Mr. Brumbaugh 16 Revised Summer 2016 Data: Treatment 2: Situation: Group Number Standing heart rate (beats/min) Reclining heart rate (beats/min) Peak heart rate upon standing (beats/min) Initial standing heart rate just before step test (beats/min) Heart Rate Average after step test (beats/min) Recovery time (seconds) Endurance (beats/min) Heart Rate 1 2 3 4 5 6 Fitness Points 1 2 3 4 5 6 Total fitness points Average total fitness points for treatment 2 Arbitrary Fitness Level Table 6B Treatment #2 Data Table Record your heart rate and fitness points on this table captured from Treatment #2 subject. Miscellaneous Notes and Observations: Record the Biology& 100 Laboratory Manual Mr. Brumbaugh 17 Revised Summer 2016 Graphing the Data: 1. Read carefully “Graphing of Data” in Appendix A, obtain a piece of appropriate graphing paper, and then construct a graph that will assist you in interpreting the results from this investigation: Graph total fitness points for subjects 1-6 for treatment 1. Graph total fitness points for subjects 1-6 for treatment 2. Graph average total fitness points for treatment 1. Graph average total fitness points for treatment 2. Appropriately label the graph with a figure number, title, and descriptive sentence. 2. Summarize the trends in fitness displayed on your graph by referring to the data displayed on your graph (Refer to a data by figure number). 3. Construct an additional graph to reveal some other trend from your research. For example compare recovery time, heart rate before and after exercise, or heart rate after exercise of each treatment. Use the graphing information to assist your design (Appendix A). 4. Summarize the trends in fitness displayed on your graph by referring to the data displayed on your graph. (Refer to a data by figure number). Biology& 100 Laboratory Manual Mr. Brumbaugh 18 Revised Summer 2016 Conclusions: 1. Does the data support or refute the hypothesis proposed by the class? Explain using data from the experiment (Refer to a data by figure number). 2. Using your data (Refer to a data by figure number), are there additional conclusions one could draw from this experiment? 3. Explain why an experiment has only one independent variable, and identify the independent variable for this experiment? 4. How could this experiment be improved to get results that would allow the formulation of more valid conclusions? Give specific ways the experiment could be improved! Biology& 100 Laboratory Manual Mr. Brumbaugh 19 Revised Summer 2016 Application Questions: 1. When subjects moved from a standing position to a reclining position, how did their heart rate change, by how much, how do you account for this change, and was the result what you predicted? (Refer to a data by figure number) 2. When subjects moved from a reclining position to a standing position, how did their heart rate change, by how much, how do you account for this change, and was the result what you predicted? (Refer to a data by figure number) 3. Why does research indicate that most heart attacks occur as people get out of bed after a night’s sleep? 4. Why would athletes need to work longer and harder before their heart rates were at the maximum value? Biology& 100 Laboratory Manual Mr. Brumbaugh 20 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: . Heart Rate & Fitness Exercise Name: . Before coming to lab carefully read the previous pages on the Principles of the Scientific Method then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. 1. Restate the following hypothesis in an “If-Then” statement. Hypothesis: Students that study twohours outside of class for every one-hour in class usually get better grades than students that study half that amount of time. 2. Identify the independent and dependent variables in the following experiments: Pea plant height measured daily for 30 days. Dependent variable: Independent variable: Number of leaves found on pea plants 5 days after having been treated with gibberellic acid. Dependent variable: Independent variable: 3. Suggest a control treatment for each of the following two experiments: Pea plants are sprayed with 5ml. aqueous solution of gibberellic acid and their height determined daily after the spraying. Control treatment: Pulse rate is determined after 3 minutes of aerobic exercise. (Hint: the control is what the heart rate after exercise will be compared to.) Control treatment: Biology& 100 Laboratory Manual Mr. Brumbaugh 21 Revised Summer 2016 4. Should the data obtained from the following experiment be plotted as a line graph or a bar graph? Briefly explain your reasoning: Pea plant height measured daily for 30 days. (See Appendix A for help.) Line graph or Bar Graph (circle one) Explain why you came to this conclusion? 5. Write a question, a hypothesis, and identify the independent, dependent and three control variables that you would like to investigate in this laboratory (heart rate experiment). See pages of the Perspectives for help. Biology& 100 Laboratory Manual Mr. Brumbaugh 22 Revised Summer 2016 Laboratory 2: Microscopic Observation of Cells Pre-lab Assignment Before coming to lab carefully read the following pages on Microscopic Observation of Cells plus Appendices B and D, then answer the pre-lab questions, pages 37 & 38. Perspectives The microscope is one of the principal tools of the biologist. Without the microscope, many of the great discoveries of biology would never have been made. The light compound microscope (Figure 1) is the type of microscope most commonly used. Proper, comfortable use of the instrument demands practice. The practice afforded you in this exercise depends upon familiarity with the parts of the microscope and with their interactions. Note each of the following features of the microscope and their individual uses allow you to take full advantage of the use of a microscope. Ocular: These contain lens that magnify (usually 10x) the specimen. Revolving Nosepiece: Device used to change magnifying lenses (objectives). Objective Lens: Magnifying lenses usually ranging from 4x to 100x. Sub-stage Condenser: This adjustable device gathers the light rays from the light source and focuses them onto the specimen stage. Iris Diaphragm: This lever adjusts to control the amount of light shown onto the specimen. Coarse and Fine Focus Knobs: These adjustable knobs are used to focus the specimen. The course focus knob only needs to be used with the 4x objective. Figure 1 Compound Light Microscope A typical compound light microscope used in many biology labs. Magnification and Resolution In using the microscope it is important to know how much you are magnifying an object. To compute the total magnification of any specimen being viewed multiply the power of the eyepiece Biology& 100 Laboratory Manual Mr. Brumbaugh 23 Revised Summer 2016 (ocular lens) by the power of the objective lens being used. For example, if the eyepiece magnifies 10x and the objective lens magnifies 40x, then 10 times 40 gives a total magnification of 400x. The compound microscope has certain limitations. Although the level of magnification is almost limitless, the resolution (or resolving power) is not. Resolution is the ability to discriminate two objects close together as being separate (clarity). The human eye can resolve objects about 100 µm apart (note: 1 µm = 1 micrometer = 1 millionth of a meter). Under ideal conditions the compound microscope has a resolution of 0.2 µm, about 500 times the resolving power of the human eye. Objects closer than 0.2 µm are seen as a single fused image. Resolving power is determined by the amount and physical properties of the visible light that enters the microscope. In general, the greater the amount of light delivered to the objective lens, the greater the resolution. The size of the objective lens aperture (opening) decreases with increasing magnification, allowing less light to enter the objective lens. Thus, it is often necessary to increase the light intensity at the higher magnifications. Depth Perception and the Microscope Any microscopic object viewed has depth as well as length and width. While the lens of your eye fully adjusts to focus on an object being viewed and provides an image that allows your brain to develop a three dimensional interpretation, the lenses of a microscope are focused mechanically and can only “see” in two dimensions, length and width. For example, if the specimen you are examining has three layers of cells, you will only be able to focus on one cell layer at a time. In order to perceive the relative depth of your specimen, use the fine adjustment knob to focus through the different planes (i.e. the three cell layers) individually to build a three-dimensional picture or interpretation of your specimen. The Field of View and Estimating the Size of Specimens When you view an object under the microscope you will observe that it lies inside a circular field of view. Each magnification lens has a different sized field of view. If you determine the diameter of the field of view you can estimate the size of an object seen in that field. As you increase the magnification, the field of view (and diameter) gets proportionately smaller. As a consequence, a critter that appears small under scanning power (4x) may appear large under high power. The actual size of the critter did not change, only the space in which you placed it for viewing. The Oil Immersion Lens Although the oil immersion lens (100x) when used properly offers the ability to view objects at high magnification, the objects viewed in this lab exercise do not warrant its use. As its name implies, an oil immersion lens requires a drop of immersion oil to be in contact between the lens and the slide for the lens to function effectively. Since immersion oil has the same refractive index as glass, it prevents the scattering of light as light passes from the glass slide to the objective lens (also made of glass). Poor resolution is the result if the oil immersion lens is used without oil since light will be bent (and thus scattered) as it passes from the slide to air, and then through the oil immersion objective lens because air and glass bend light differently as a result of having different refractive indexes. Care of the Microscope: Your microscope is an expensive instrument that must be given proper care. Always follow these general instructions when using a microscope. 1. Carry the microscope with both hands, one hand under the base, and the other on the arm. When getting ready to put the microscope away, always return it to the low power or scanning power objective over the stage, lower the stage to its lowest position, and wrap the cord securely around the light source and the base of the arm. 2. When setting the microscope on a table, always keep it away from the edge. 3. It is generally best to clear your lab table of items that are not being used. Biology& 100 Laboratory Manual Mr. Brumbaugh 24 Revised Summer 2016 4. The lenses of the microscope cost almost as much as all of the other parts together. Never clean them with anything other than lens paper. Paper towels and other paper tissues will scratch the lens. 5. Please inform the instructor or the biology lab technician of any microscope damage or irregularity in its operation as soon as possible. Do not return a faulty microscope without first informing the instructor or lab tech. 6. You are responsible for the microscope while you are using it. Treat it with care! Exercise: Microscope Function and Uses Goals of this Lab Exercise Identify the parts of a compound light microscope and use a microscope to competently examine biological specimens Determine the diameter of the field of view for the various objective lens of a microscope Accurately sketch, describe, and cite the major functions of the structures and organelles of the cells examined in this lab exercise Estimate the size of specimens when viewed with a microscope Learn to use metric units correctly when measuring specimens on the compound microscope Introduction 1. Techniques for Setting Up & Viewing Objects with a Compound Microscope. Be familiar with the following procedures outlining the correct usage of the microscope before coming to lab. The steps that follow should be observed in this lab exercise and all other microscope lab activities in this and other courses/lab experiences. A. Place the low-power objective (4x or scanning) in position over the stage by grasping the nose piece and NOT the objectives. In changing from one objective to another, you will hear or feel a click when the objective is set in the proper position. B. Make certain that the lenses are clean. Dirty lenses will cause a blurring or fogging of the image. The high power and ocular lenses are the lenses that most often get dirty. To clean a microscope lens: 1) Place a drop of lens cleaning fluid on a piece of lens paper; 2) Clean lens with a gentle circular motion; 3) Finally dry the lens with a fresh piece of lens paper and 4) Dispose of lens paper in the trash. Always and only use lens paper for cleaning a microscope lens! Any other material (including Kim wipes or your finger) may scratch the lens. C. Plug in the electrical cord to turn on the sub-stage light, turn power switch to on, and adjust the light control (rheostat) to dim the light to just illuminate (pale yellow) the viewing field. While looking through the oculars (use both eyes for ease of microscope operation) turn the light control until the light in the field of view is an evenly lit white field. D. Position the condenser as high as it will go by turning the sub-stage adjustment knob. As you view a specimen you will have to lower the condenser slightly to set the condenser at the proper level. By raising or lowering the condenser, you can change the contrasting effects of the microscope to increase the ability for you to view the specimen with more resolution. E. Open the iris diaphragm by means of the black lever attached to the condenser apparatus, which is found just below the stage of the microscope. 2. Getting Started Viewing Specimens Using the Compound Microscope. A. Selecting the objective to begin. a. If you need to scan the slide to find the location of a specimen use the low power scanning objective (4x) with its larger field of view. b. If you have a pretty good idea where the specimen is located on the slide it is okay to start with the medium power objective (10x). Biology& 100 Laboratory Manual Mr. Brumbaugh 25 Revised Summer 2016 B. C. D. E. F. c. Because of the danger of damaging these lenses with their very small field of view and shorter focal lengths, never begin microscopic examinations with the high power (40x) or the oil immersion (100x) objectives. d. See the previous page for information concerning the oil immersion lens. Place the slide on the stage for viewing. a. Make certain that the low power scanning objective (4x) or the medium power objective (10x) is “clicked” properly in place. b. Lower the stage away from the objective with the coarse adjustment knob. c. Place a properly prepared slide on the stage and secure with the stage clips or mechanical stage depending upon which is present on your microscope. d. Viewing from the side of the stage move the part of the slide with the object to be viewed directly above the brightly illuminated sub-stage condenser. Proper Focusing Technique for low scanning (4x) and medium power (10x) objectives. a. Viewing the stage from the side, use the coarse adjustment knob to raise the stage until the stop is reached that will prevent further movement of the stage. b. Looking through the eyepiece (ocular, usually 10x power) lower the stage slowly by turning the coarse adjustment knob away from you until the object is in focus. It should take less than a quarter of a turn to bring the image into focus. c. Use the fine adjustment to bring the object into sharp focus. d. Adjust the amount of light with the iris diaphragm and intensity of light with the condenser for optimum viewing. Too much or too little light adversely affects the contrast of the image viewed! Tips! a. When it is difficult to find a specimen to focus on (e.g. when examining cheek cells), bring the edge of the cover-slip into the center of the field of view, and then try focusing on the edge, then search the slide for the desired specimen. b. Reduce the light intensity (can increase contrast) to aid in the observation of viewing clear/transparent objects such as amoeba or cheek cells. c. To avoid eyestrain practice keeping both eyes open. Many biologists are capable of observing a specimen and sketching it at the same time! Try it out in today’s lab. Switching from Lower to a Higher Power Objective. a. First, be sure the object that you want to view at a higher magnification is in the center of the field of view (Why?) and sharply focused under low power. b. Switch to a higher power objective and watch from the side to make sure that the objective lens does not touch the slide. Since most microscopes are par focal (meaning that the focal plane of each lens is matched to be reasonably closely related) means that little refocusing is needed when moving from one lens to another. Only fine adjustment may be required. If properly focused at low power, and the slide is prepared correctly (i.e. the specimen is thin and flattened by a cover slip), you should be able to switch automatically from low to high power without fear of having the high power objective lens scraping or touching the slide. c. The object should be in focus, or almost in focus (use only the fine focus knob). Re-Focus with the Fine Adjustment under High Power (40x). a. Only use the fine adjustment focus knobs at high power! To avoid damaging the lens, never use the coarse adjustment when the high-power (40x) or oil immersion objective (100x) is in place. b. The working distance is the distance between the specimen viewed and the objective lens of the microscope. As you increase magnification the working distance becomes less and less. The objective will be almost touching the cover slip when properly focused at high power Biology& 100 Laboratory Manual Mr. Brumbaugh 26 Revised Summer 2016 G. Adjust the amount and intensity of light for optimum viewing by using the iris diaphragm or condenser. The amount of light may need to be increased since less light passes through the high power objective at higher magnification. Do not turn or move the chrome screws under the stage attached to the condenser apparatus as these either hold or adjust the condenser alignment. H. Removing the slide from the stage. a. Switch the objective to low power (4x). Removing a slide while under high power may scratch the lens. b. Carefully lower the stage using the coarse adjustment knob, open the stage slide holding mechanism, and carefully remove the slide. I. Disposal of Wet Mounts. a. Discard the cover slip (plastic cover slips in the trash, glass cover slips in the broken glass container at the front of the lab). b. Wash the slide with soap and water and then rinse the slide with deionized (dH2O) water at the sink in the back or place into the beaker containing slide cleaning solution and skip step c. c. Place the slide to dry on the paper towel labeled “Wet Clean Slides” on the drain board next to the large stainless steel sink at the front of the room. J. Prepared slides. a. Return to their proper location within the plastic slide container on the lab supply cart 3. Preparing a Wet Mount. A. Place a drop of water on a clean slide with a dropper. B. Put the object in the water drop. C. Lower one edge of the cover-slip to the edge of the water drop as shown in the illustration (Figure 2). Lower the cover-slip slowly to avoid air bubbles. A gentle tapping will usually remove any bubbles that may be present. Blot any excess water with a paper towel. More water can be added with a dropper at the edge of the cover slip. Do not let your specimen dry out. Figure 2 Wet Mount Preparation The critical aspect of this technique is the amount or water that is used to make the slide. Materials Common Use Items on the Center Table Lens Paper and Lens cleaning solution Metric Rulers Clean microscope slides Cover slips Eye droppers Bottle of deionized water (dH2O) Forceps (tweezers) Scissors Safety Goggles Procedure As a group answer the questions on the report sheet and turn in one packet next week at the start of the lab. Answer the questions on your Report Sheet (starting on page 33) as you follow the Biology& 100 Laboratory Manual Mr. Brumbaugh 27 Revised Summer 2016 procedures outlined below. Perform procedures that can only be done in the lab before doing procedures or answering questions that can be performed outside of the biology laboratory! Part A: Observation of a Newsprint Letter In this part you will learn to use the microscope to examine a familiar object, a self-prepared slide of a newsprint letter. Refer to the previous sections to prepare your slide and observe it with the microscope. Practice adjusting your microscope to become proficient in locating a specimen, focusing clearly, and adjusting the light (via the iris diaphragm, the condenser, or the light rheostat) for optimum viewing. Materials needed for Part A Microscope slide and cover slip Newsprint letter Compound light microscope Safety Goggles Procedure: 1. Make a wet mount (Wear Safety Goggles) of a lower case letter which has been cut from newsprint (Don’t use the letter o. Why?). When placed on the microscope stage the letter should be in an upright position when viewed with the naked eye (i.e. not looking through the eyepiece of the microscope) from behind the arm of the microscope. 2. Observe this under low (4x), medium (10x), and high dry power (40x). 3. Answer questions 1-10 on the Report Sheet (pages 33 & 34) as you observe the letter with a compound light microscope. Part B: Defining Resolution and Determining the Total Magnification To be able to compare data with other scientists, microscopists need to know the structure and function of the various cellular components. Reliability of size relationships is critical. The following exercise part is designed to give the student information about the magnification of the compound light microscope. Materials needed for Part B Compound light microscope Procedure: 1. Determine the total magnification of the low, medium, high, and oil immersion fields of view. Record the data in the first two rows of Table 1 (page 34). Part C: Determining the Size of the Microscopic Field of View You will often want to know the size of the objects you are observing under the microscope. Because these objects are usually too small to permit direct measurement, it will be convenient for you to learn a method to indirectly measure them. Estimating the diameter of the field of view for the different objective magnifications will enable you to determine indirectly the approximate size of objects viewed under the microscope. Materials needed for Part C Compound light microscope Plastic Metric Ruler Procedure: The diameter of the field of view for low power is given to you in Figure 3. To verify this diameter, do the following: 1. Be sure the low-power objective (4x) is in position. Place the graduated edge of a plastic metric ruler across the midline (diameter) of the field of vision (Figure 3). Bring the ruler into focus under low power (4x). Biology& 100 Laboratory Manual Mr. Brumbaugh 28 Revised Summer 2016 2. Record the diameter in millimeters onto Table 1 on your Report Sheet ( page 34) and convert the diameter in micrometers. One millimeter equals 1000 micrometer: 1 mm = 1000 µm (Appendix B). The most common unit of measurement in microscope work is the micrometer, µm. 3. Once you have determined the diameter for the field of view at low power you can calculate the diameters of the fields of view for the rest of the objective lenses (Figure 4). For example, to calculate the diameter of the high-power field knowing the diameter of the low-power field, first find the number on each objective that indicates the magnification. Divide the high-power by the low-power magnification to get a factor that indicates how much smaller the high power field is compared to the lower power lens. For example, if the low-power objective reads 15x and the high-power 45x, dividing 45 by 15 gives a factor of 3. If the diameter of the low-power field were 1.5 mm, then the diameter of the high-power field would be 1.5 divided by 3 or 0.5 mm. Fill in the last two rows of Table 1 (page 34) on your Report Sheet. 4. Once you know the field of view diameter, you should be able to estimate the size of any organism found within that field. 1 mm = 1000 µm 1 µm = 0.001 mm Field of View App. 4.5 mm Metric Ruler (mm) Figure 3 Microscope Field Diameter Measurement This figure shows the hypothetical determination of the diameter of the field of view of a scanning or low power (4x) objective using a metric ruler. Figure 4 Magnification Conversion Table This information is used to calculate the field of view by using conversion factors of par focal objective lenses. Biology& 100 Laboratory Manual Mr. Brumbaugh 29 Revised Summer 2016 Part D Depth Perception It is important to remember that by using the coarse and fine adjustments you bring the microscope into focus at many different levels. At each setting you can see clearly only one plane of the object. To see other planes clearly, you must change the focus. Materials for Part D Nylon panty hose (~1 cm square) Prepared slide of crossed threads Compound Light Microscope Safety Goggles Bottle of deionized water (dH2O) Cover slip and slide Procedure: 1. Obtain a small piece of nylon hose approximately 1-cm square. Prepare a wet mount (Wear Safety Goggles) of this material (make the nylon piece as smooth as possible on the slide). Examine under low and high power. Notice as you rotate the fine adjustment knob back and forth slowly, you can see the fibers at different depths. You get a three-dimensional (i.e. length, width and depth) interpretation of the nylon. 2. Describe your impression of microscopic depth of field by answering question #12 on page 34. 3. Obtain a prepared slide with three colored cross threads (or make a wet mount if a prepared slide is not available.) 4. Focusing with a medium power objective (10x), locate the point where the three threads cross each other. Using proper focusing technique as outlined in the beginning of this lab determine which thread comes into focus first. Use this information to help answer question #13 on your Report Sheet (page 34). Part E: Wet Mounts and Drawings In the circles provided in the report sheet make neat and accurately labeled drawings of the cells listed below. Your sketches should be accurate (Use textbooks and Appendix D for assistance) enough so that an informed individual could identify the cells or organisms from your drawings. Mammalian wet mount: Human Cheek cells Materials for this exercise Flat-tipped toothpick Dropper bottle of methylene blue Caution! Compound Light Microscope Bottle of deionized water (dH2O) Safety Goggles Cover slip and slide Place all used toothpicks into the autoclave bag at front counter. Procedure: 1. Wear Safety Goggles for this part of lab! Place a tiny drop of deionized water in the center of the slide. Using the flat end of the toothpick, gently scrape the inner lining of your cheek. Agitate the end of the toothpick containing the cheek scrapings in the drop of dH2O. 2. Add a tiny drop of methylene blue stain to the preparation and stir again with the toothpick. Cheek epithelial cells are nearly transparent and thus difficult to see without the stain, which colors the nuclei of the cells and makes them look much darker than the cytoplasm. 3. Add a cover slip and observe under low and then under high power. Although the cells form a solid sheet of cells in your mouth, the scraping of the toothpick probably caused the cells to separate from each other. Try to find a cluster of two or three cells whose shapes have not been totally distorted. Avoid observing clumps of cells that show little cellular detail. 4. In the appropriate space on your Report Sheet (page 35), use a sharp pencil to make an accurate sketch of a cluster of two or three cells as viewed at high dry power (or one cell if 2 or 3 cells can’t be found together). Estimate and record the approximate size (in micrometers, µm) of a single epithelial cell in micrometers and attach a drawing scale. Biology& 100 Laboratory Manual Mr. Brumbaugh 30 Revised Summer 2016 5. Identify, neatly label, and describe the general cellular function for the following cellular structures: Plasma Membrane, Cytoplasm, and Nucleus. Filamentous algae wet mount: Spirogyra Materials for this exercise Filamentous algae (Spirogyra) Cover slip and slide Compound Light Microscope Pipette or dissecting needle Safety Goggles Procedure: 1. Wear Safety Goggles for this part of lab! Prepare a wet mount of Spirogyra: Use a pipette or a dissecting needle to place a sample of the freshwater algae, Spirogyra, on a clean slide, cover with a cover slip, and observe with low and high power. Use a dissecting needle to straighten and untangle the strands of Spirogyra on your microscope slide. 2. In the appropriate space on your Report Sheet, use a sharp pencil to make an accurate sketch of two or three consecutive cells of Spirogyra as viewed at high dry power. 3. Estimate and record next to your drawing the approximate length and width (in micrometers, µm) of a single cell and attach a drawing scale. 4. Identify and neatly label on your sketch the following structures. Use your textbook as a reference if needed. Cell Wall: Often coated with slime in filamentous algae such as Spirogyra. Cytoplasm: the internal aqueous environment inside the cell. Nucleus: Embedded within the cytoplasm of Spirogyra; Very light in color and therefore difficult to find if the diaphragm is opened too wide and/or the condenser is not adjusted properly. Chloroplast: In Spirogyra, it appears as a long spiral band that runs the length of the cell. Vacuole: Clear and large membrane-bound organelle within the cell that stores the sugars produced by photosynthesis. Being clear it is difficult to observe in Spirogyra cells unless the diaphragm is used to decrease the light intensity and the condenser and fine adjustment are properly adjusted. Microscopic organism of your choice: Pond Water “Scum”. Materials for this exercise Pond water and other cultures of microorganisms Cover slip and Depression slide Compound Light Microscope Safety Goggles Procedure: 1. Wear Safety Goggles for this part of lab! Prepare a wet mount of pond water or of other organisms provided by your instructor by using a depression slide and cover slip, and observe using low- and high-power. 2. In the appropriate space on your Report Sheet draw (with the magnification and a size scale) at least two organisms of your choice as viewed at the high power or at the highest magnification possible that allows you to view the whole organism. 3. Neatly label all cell parts as above and describe your critters means of locomotion. 4. Use the identification keys in the lab to identify the kingdom (and phylum, if possible) that the organism belongs to. 5. Estimate the length and width (or diameter) of this organism in µm. Record these dimensions on your drawing and attach a drawing scale. 6. Turn in pages 33 through 35 as a group to include all drawings, magnifications, and scales. Biology& 100 Laboratory Manual Mr. Brumbaugh 31 Revised Summer 2016 Biological Doodling Space Biology& 100 Laboratory Manual Mr. Brumbaugh 32 Revised Summer 2016 Report Sheet Lab Section: . Microscopic Observation of Cells Exercise Group Names: Note: . . . . Perform procedures that can only be done in the lab before doing procedures or answering questions that can be performed outside of the biology laboratory! 1. Using low-power (4x) compare the position and orientation of the image of the letter as seen through the ocular with the position of the letter as seen on the slide without using the microscope. What two orientation differences of the image are there when compared to the image seen without a microscope? In the circle below use a sharp pencil to make a simple sketch of the letter as viewed under lowpower. Letter viewed at magnification x 2. While looking through the oculars slowly move the slide away from you by using the stage control knobs. Which way does the image move? (Place answer here) 3. While looking through the oculars, slowly move the slide from right to left using the stage control knobs. Which way does the image move? (Place answer here) 4. Make a rough estimate of how much of the letter is visible when viewed under high power? (Give a percent based on the differences in the size of the field of views) (Place answer here) 5. Does the switch from low power (4x) to high power 40x) change the position of the image? (Place answer here) Biology& 100 Laboratory Manual Mr. Brumbaugh 33 Revised Summer 2016 6. Why is it necessary to center your object (or the position of the slide you wish to view) before changing to high power (40x)? (Place answer here) 7. Under high power (40x) is the illumination brighter or less bright than it is with low power? (Place answer here) 8. Move the iris diaphragm lever in each direction while observing the field. What happens? (Place answer here) 9. Is it more desirable to increase or decrease the light when changing to a higher magnification? (Place answer here) 10. What is the approximate actual height in millimeters and microns (µm) of your letter? Height = mm = µm (Place answers here) 11. Fill in Table 1. Low Power Magnification of Objective Lens Total Magnification Field Size (diameter) Field Size (diameter) Medium Power High Power Oil Immersion x x x x x x x x mm mm mm mm µm µm µm µm Table 1 Part B and C Data Fill out this table to show the Magnification and Field Size dimensions for your microscope. 12. Space to describe your impression of microscopic depth of field and why this feature of a microscope is important to microscopists? 13. Suppose a slide were set up with Yellow, Blue, and Red threads that cross at a single point top to bottom, which thread would come into focus first if you positioned the stage as close to the low power objective lens as possible and then brought the slide into focus? Circle the letter of the correct response. A. Yellow first B. Blue first C. Red first. D. All three colors at once. Biology& 100 Laboratory Manual Mr. Brumbaugh 34 Revised Summer 2016 14. Drawings of Wet Mounts for Part E. See Appendix D for what is required in the drawings below. Cheek Cell viewed at x Drawing Scale Spirogyra viewed at x Drawing Scale . . Pond Water Organisms Organism of choice viewed at Drawing Scale Biology& 100 Laboratory Manual Mr. Brumbaugh x Organism of choice viewed at . Drawing Scale 35 x . Revised Summer 2016 Biological Doodling Space Biology& 100 Laboratory Manual Mr. Brumbaugh 36 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: . Microscope Techniques Exercise Name: . Before coming to lab carefully read the previous pages on Microscope Techniques then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. A.Why is it necessary to center your object (or the position of the slide you wish to view) before changing to high power? Complete the table below and use the data to answer questions B and C. Microscope Number 1 2 3 4 Objective Lens 25x 15x 20x 40x Ocular Lens 5x 10x 10x 5x Total Magnification B. Given that each slide had the same density of microbes, with which microscope would you expect to observe the greatest number of microbes at any given instant? Why? C. If a slide showing the same organism is examined with each of the microscopes, above, with which two microscopes will the microbe appear to move with the same degree of rapidity? Why? microscope will it appear to move the slowest? Why? D.What is meant by resolution (resolving power) of a microscope? E. After switching from one objective to another, why is it often necessary to readjust the diaphragm and the condenser? Biology& 100 Laboratory Manual Mr. Brumbaugh 37 Revised Summer 2016 Use the data in the table below for a hypothetical microscope to answer questions F and G. Objective Used Low power Medium power High power Oil immersion Total Magnification 30x 150x 300x 1500x Diameter of Field of View 6000µm F. Calculate the diameter of the field of view at medium, high, and oil immersion. Record your answers in the table above, and show and/or explain your work below. G. You observe an object whose length is 1/4 the diameter of the medium power field of view of the hypothetical microscope in the table above. What is its length in microns? ____________ in millimeters, mm? _______________. Show and/or explain your work below. Biology& 100 Laboratory Manual Mr. Brumbaugh 38 Revised Summer 2016 Laboratory 3: Transporting Across Boundaries Pre-lab Assignment Before coming to lab carefully read the following pages on Transporting Across Boundaries then answer the pre-lab questions, page 51, and define the terms at the end of the perspectives section. Be prepared to hand in your responses to the pre-lab questions and the definitions at the start of lab. Perspectives A good way to think about the movement of substances into or out of a cell is to envision the system that you are studying as being different compartments separated by a membrane barrier that is permeable (allows movement across) to certain substances and impermeable (allows no movement across) to other substances. The substances are referred to as the solute and the dissolving solution (usually water) is referred to as the solvent. In these experiments the barrier (either a dialysis sac or cell membrane) acts as a semi-permeable membrane. Your task is to determine what solute molecules are allowed to move across the membrane (permeable) or not (impermeable). The movement of solute across a semi-permeable membrane can either be an active transport process (requiring the use of energy in the form of ATP) or a passive process (requiring no energy). This lab will investigate passive processes. Smaller, non-polar molecules can move across a membrane by passing between the phospholipid molecules of the membrane (called simple diffusion). While smaller, polar molecules, and ions (charged atoms) can move through specific protein pores embedded into the membrane (called facilitated diffusion). Both of these routes are classified as passive transport because they require no energy input by the cell. The mechanisms or forces that drive this process are Brownian motion (random molecular movement) and a favorable concentration gradient (more of one solute on one side of the membrane than that same solute on the other side). If all other factors are constant, then eventually the solute will come to equilibrium (balance) across the membrane. Osmosis is a special term that describes the movement of a solvent (usually water) across a semipermeable membrane by diffusion through protein channels called aquaporin. The force that moves water, though, is not the water concentration, but rather the solute concentration across the membrane. The solute is termed an osmotically active substance (OAS), and can be any molecule or matter dissolved in the solvent. The trick is to measure the total OAS and then to measure the solutes that are able to move across the semi-permeable membrane (remember they will reach their own equilibrium). By comparing the difference between the permeability of the OAS, you can then predict the OAS concentration at equilibrium and be able to predict the net movement of water. Three terms are used to describe the relationship between OAS concentration across a membrane: hypertonic, hypotonic, and isotonic. A hypertonic condition would have more OAS outside the cell, a hypotonic condition would have more OAS inside the cell, and an isotonic condition would have a balance of OAS across the membrane. Because of the differences or similarities of concentration, water will move to the area of higher OAS and could cause radical changes in the shape of the cell. Hypertonic solutions cause the cell to lose water and shrink in size or crenate; hypotonic solutions cause the cell to gain water and expand or potentially burst (lysis), while isotonic has no net movement of water. How would changing a cell from solution to solution affect cell function? The key to understanding this exercise is to figure out 3 things. 1. What concentration gradients you have created? 2. In which direction will the net flow of given molecules travel within their concentration gradient or in which direction will the net flow of molecules within their concentration gradient try to flow? 3. To which type of molecule is the membrane permeable? Biology& 100 Laboratory Manual Mr. Brumbaugh 39 Revised Summer 2016 Define each of the following terms on a separate sheet of paper: solvent, solute, precipitate, semi-permeable membrane, passive transport, active transport, concentration gradient, osmosis, osmotic concentration, osmotically active substance, simple diffusion, hypertonic, hypotonic, isotonic, and transport proteins. Exercise: Transporting Substances across Membranes Goals of this Lab Exercise To understand the mechanisms used to move substances across membranes Understand the factors that can influence rates of transport across membranes Define terms associated with moving substances across membranes Apply the principles of moving materials across membranes to medical or environmental issues Introduction Through a series of demonstrations and exercises, the principles of moving substances across membranes will be studied. Each exercise will reveal a different yet related aspect of transportation into and out of cells. Bear in mind that these principles are put into practice continually by cells trying to function and maintain homeostasis in an ever changing environment. Whether that environment includes existing as a single celled protistan, like a euglena, or a multi-celled animal, like a homo sapien, each cell needs to practice the principles for moving molecules into or out of its cellular body or run the risk of cellular death or at the minimum, disruption of cellular function. Each group will gather data to complete Part A, three members will complete Part B, and one member will complete Part C. Share the data from all parts and turn in the Report Sheet next lab as one group. Procedure Part A. Demonstrations of Molecular Movements through Different Media The following demonstrations are used to show the process of molecular movements via Brownian motion. The first is movement through a semi-permeable membrane, the third is through a semi-solid (agarose) medium, and finally through a living membrane. Write hypotheses for each demonstration, gather the data of the molecular movements during lab, record on the Pre-Lab Report Sheet page 51. Demonstrations Students will not set-up the following demonstrations but will be required to write hypotheses before coming to lab on page 51, and take measurements for each demonstration. Thistle-tube demonstration. The dialysis tubing, which is a semi-permeable membrane, contains a concentrated sugar solution and a blue dye. This sac was then lowered into a beaker filled with water only. What principle does this demonstration illustrate? The petri dish filled with agarose gel to which two different dyes were added. This represents the third demonstration of movement through a semi- solid media. Measure the halo distance of movement for each dye as it moves through the media. Are there differences in the rate and total movement of each dye and why? The third demonstration shows the results after potato slices were left in different solutions for about one day. Feel the slices in each watch plate and explain what could have happened to the potato slices Biology& 100 Laboratory Manual Mr. Brumbaugh 40 Revised Summer 2016 Answer questions 1 through 3 pertaining to the demonstrations on the Report Sheet page 45 and turnin as a group. Part B. Diffusion through a non living membrane The purpose of this part is to demonstrate the action of a differentially permeable non-living membrane. Such action can then be applied to the study of transport mechanisms in living cells. Section 1: The Set Up Materials 1 small funnel laboratory balance four moistened dialysis sacs 5% I2KI (Lugol’s) solution 1 – 10 ml. graduated cylinder pieces of string 40% glucose solution 10% Starch solution wax pencil and scissors 2 - 400 ml. Beakers 10% NaCl solution Safety Goggles Procedure: 1. Formulate a hypothesis about the movement of the various solutes across the dialysis sac. Write them down on page 46. 2. Wear Safety Goggles when transferring reagents from container to container. 3. Obtain two 400ml glass beakers and number them 1 and 2. Fill beaker 1 half full with dH2O (the toggle switch faucet at the back sink) and fill beaker 2 half full with Lugols’ (5% I2KI) iodine solution. 4. Obtain 2 moistened cellulose dialysis tubing lengths (Sacs 1 and 2). To open the cellulose tubing rubs one end between your thumb and forefinger and place a funnel into the open end. Fold over the other end of the tube (about 1 cm.) and tie with string in the middle of the folded area (Figure 1). 400ml beaker dH2O or 5%I2KI Glucose and NaCl or Starch Closed Dialysis Tube Figure 1 Dialysis Tubing Set-up Diagram for the investigation of the permeability of dialysis tubing. 5. To Sac 1 using a graduated cylinder and a funnel add about 5 ml of 40% glucose solution followed by about 5 ml of the salt solution. Close the open end of the sac by pressing out the air, folding over the open end, and tying it securely with string to form a closed leak proof sac. Carefully remove excess string and tubing from each end of the sac (Why?). 6. To Sac 2 using the graduated cylinder and funnel after rinsing with dH2O, add about 10ml of 10% starch solution. Close the open end of the sac by pressing out the air, folding over the open end (about 1 cm.), and tying it securely with string to form a closed leak proof sac. Carefully remove excess string and tubing from each end of the sacs (Why?). 7. Rinse the sacs under running tap water to remove any solution from the outside of the sacs and carefully blot the sacs dry (Do not squeeze sacs!) with a paper towel. Biology& 100 Laboratory Manual Mr. Brumbaugh 41 Revised Summer 2016 8. Determine the mass of the sacs in grams by using a weighing boat on a laboratory balance that can be found on the side counters. Place your sac into the plastic weigh boat and zero the scale before weighing your sample. Record the weights in Table 1 (page 45) 9. Place Sac 1 into the beaker with the dH2O and Sac 2 into the beaker with the Lugol’s iodine solution. Start both sacs at the same time and note the time. 10. Allow the sacs to remain undisturbed in the beakers for 1 approximately hour. After one hour you will remove each of the sacs, gently and carefully blot them dry, weigh them, record the data in Table 1 of the Report Sheet (page 45), and begin Section 2 the Chemical Analysis. Section 2: The Chemical Analysis Materials Test tube rack with 4 test tubes 600 ml beaker with boiling chips Small plastic beaker and scissors test tube clamps Benedict’s solution Safety Goggles hot plate silver nitrate (AgN03) Bottle of dH2O and Transfer pipettes Procedure: 1. Benedict’s test for the presence of sugar in the Solutions. Wear Safety Goggles! Caution! Benedict’s Reagent is very caustic. It can burn holes in clothing and digest skin!! Wear goggles for eye protection! Clean up spills immediately after first neutralizing with vinegar. If spilled on your skin (it feels slippery and begins to burn after several minutes) wash thoroughly with tap water. Report all accidents to the instructor. 2. Label three clean test tube: control, sac, and beaker and add five drops of Benedict’s reagent to each test tube. 3. Use scissors to cut off one end of the sac 1 and drain into a small plastic beaker. 4. Add five drops of dH20 from the dropper bottle to the “control” test tube . 5. Use a clean disposable pipette, put 5 drops of sac 1 solution in the “sac” test tube. 6. Use a clean disposable pipette to put 5 drops of beaker 1 solution in the “beaker” test tube. 7. Heat all three test tubes in a boiling water bath for about 2-3 minutes. 8. Record the colors and intensity of the colors of the solutions in your Table 2 (page 45). 9. Add five drops of the “beaker” water to a test tube and with a clean disposable pipette then add five drops of AgNO3 (silver nitrate). Note the results in Table 2. 10. Note the presence or absence of starch in the sac 2 and the beaker 2 by noting any color changes in Table 2. 11. Answer questions 1 through 9 on pages 46 and 47 on the report sheet and turn in a as a group. Part 3: The Clean up Procedure: 1. Solutions from the beakers may be poured down the sink. 2. Place used dialysis tubing in the wastebasket or taken home as a gift from GRC. 3. When finished with the hot plate turn it off and unplug it. Leave the water and boiling chips in the 600 ml beaker on the hot plate to cool. 4. Empty the test tubes with Benedict’s and AgNO3 solutions into the waste container near the back sink. 5. Clean the test tubes using detergent, water, and a test tube brush. Rinse with dH2O and place the cleaned tubes upside down in a test tube rack on a paper towel on your table to dry. 6. Rinse and clean using soap and water all other glassware used, rinse with dH2O, and place them on a paper towel on your table to dry. 7. Return all other materials (i.e. wax pencils, scissors, etc.) to their proper locations. Biology& 100 Laboratory Manual Mr. Brumbaugh 42 Revised Summer 2016 8. Wipe down the surface of your lab counter and the common table with detergent and tap water to clean up any solutions that might have leaked or spilled. Part C. Diffusion of a solvent through a living membrane = Osmosis The purpose of this part is to demonstrate the action of a “living” semi-permeable membrane. How do things pass through a cells membrane? What precautions must a cell take to ensure homeostasis and maintain functions, while still being an active vibrant cell? Think of how the principles demonstrated here can be applied to the normal and abnormal environments cells can find themselves involved with and yet still can maintain their lives. Remember to formulate hypotheses for each situation in the following part of this exercise. Solutions A, B, or C are either isotonic, hypertonic, or hypotonic. Reread the perspectives about these conditions and formulate your hypothesis based on this information Materials Compound Light Microscope Samples of Red Blood Cells Microscope slides Samples of Elodea canadensis Cover slips & Safety Goggles Solutions A, B, and C Procedure: Red Blood Cells and Osmosis 1. Write down a hypothesis for each of the conditions A, B, C and D on page 47. 2. Label four microscope slides A, B, C, and D. 3. Wear Safety Goggles! Place a drop of blood on slide A with a cover slip and observe the shape of the red blood cells. Record you observations in Table 3 row A of the Report Sheet (page 47). 4. Place a drop of solution A on slide B, add a cover slip. Place the slide on the stage and add a drop of blood to the edge of the cover slip. Capillary action should move the blood under the cover slip. Watch blood cells at the edge of the advancing blood as they meet solution A, then record your observations in Table 3 Row B. 5. Repeat step 4 for solutions B and C. 6. Wash the slides in soap and water and place them in the slide cleaning beaker by the back sink. 7. Answer questions 2 through 5 on pages 47 and 48 on the Report Sheet as a group. Procedure: Plant cells and Osmosis 1. Write down a hypothesis for each of the conditions A, B, C, and D on page 48. 2. Wear Safety Goggles! Place a piece of an Elodea canadensis leaf with a drop of water from the solution the sprigs of Elodea canadensis are being held on a microscope slide mark A and add a cover slip. 3. Place the slide on a microscope stage and observe the shape of the cells and record your observations in Table 4 row A of the Report Sheet (page 47). 4. Place a piece of the Elodea canadensis leaf on the slide marked A cover with a cover slip and add solution A to the slide and record your observations in Table 4 row B of the Report Sheet (page 47). 5. Repeat step 4 for solutions B and C. 6. Dispose of the Elodea canadensis leaves on a paper towel, let them dry out (desiccate), and toss the towel into the trash. 7. Wash the slides in soap and water and place them in the slide cleaning beaker by the back sink. 8. Answer questions 2 through 5 on pages 48 and 49 on the Report Sheet as a group. 9. Answer the additional questions on page 49 on the Report Sheet on a separate sheet of paper as a group. Biology& 100 Laboratory Manual Mr. Brumbaugh 43 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 44 Revised Summer 2016 Report Sheet Lab Section: . Transporting Across Boundaries Exercise Group Names: . . . . Part A: Demonstrations. Record your data and answer the following questions. 1. What is the principle demonstrated by the thistle-tube demo? 2. What is the principle demonstrated by the agar plates and dyes? How can you explain the differences (if any) in the movement of the two different dyes? Write your measurements in the space below 3. What is the principle demonstrated by the potato slices? Why do you think the slices felt different (if they did.)? Part B Diffusion through a non living membrane Treatment Beginning Weight (grams) Sac 1: 40 % glucose and 10% NaCl solution soaked in a beaker of distilled water Sac 2: 10% starch solution soaked in a beaker of I2KI and water Final Weight (grams) Table 1 Weights of Dialysis Tubes Beginning Weights of dialysis sacs containing various solutions and Final Weights of dialysis sacs after being soaked in various solutions for 1 hour. Sac and Test Performed Test results for sac contents (present or absent) Test results for beaker content (present or absent) Sac 1: Benedicts test for Glucose (Note and record color change) Sac 3: Silver nitrate test for NaCl (Note formation of a precipitate) Sac 4: Iodine test for Starch (Note and record color change) Table 2 Chemical Analysis of Dialysis Tubes Results of Benedicts, Silver nitrate, and Iodine Tests on the various sacs and their solutions. Biology& 100 Laboratory Manual Mr. Brumbaugh 45 Revised Summer 2016 Answer the following questions concerning the movement of molecules across a semi-permeable membrane. 1. State a hypothesis for each of the sac conditions. 2. In which situations or sacs did Net Osmosis or Simple Diffusion occur? Explain your reasoning? 3. Based on your data and your knowledge of chemical structures list the relative sizes in the order of largest to smallest of the following molecules: Glucose, Starch, NaCl, and water. 4. What part of a living cell is represented by the dialysis sac? For the next four questions use the terms diffusion and/or osmosis, hypotonic, isotonic, and/or hypertonic in your answer. 5. Considering the results of Sac 1, explain the results you observed. 6. Was there a net movement of glucose or salt in either direction in Sac 1? Why or why not? 7. Considering the results of Sac 2, explain the results you observed. Biology& 100 Laboratory Manual Mr. Brumbaugh 46 Revised Summer 2016 8. Considering the results of Sac 2, explain why there was or was not a significant change in the weight of the sac. 9. What single characteristic of the semi-permeable membrane (dialysis sac) used in the lab determines which substances can pass through them? Part C Diffusion through a living membrane Condition A (blood only) Appearance and Condition of Red Blood Cells B C D Table 3 RBC Data Observations of the potential changes in cell structure of Red Blood Cells in test solutions. 1. Write a hypothesis for each of the conditions using your experiences with non-living membranes. 2. Which of the three solutions was hypertonic to the red blood cells? Explain your answer. Biology& 100 Laboratory Manual Mr. Brumbaugh 47 Revised Summer 2016 3. Which of the three solutions was hypotonic to the red blood cells? Explain your answer. 4. Which of the three solutions was isotonic to the red blood cells? Explain your answer. 5. What conditions within the human body might lead to results similar to those you experienced here? Condition Appearance and Condition of Elodea canadensis Cells A (stock H2O) B C D Table 4 Elodea canadensis Data. Observations of the potential changes in cell structure in Elodea canadensis cells in test solutions. 1. Write a hypothesis for each of the conditions using your experiences with non-living membranes. 2. Which of the three solutions was hypertonic to the Elodea canadensis cells? Explain your answer. Biology& 100 Laboratory Manual Mr. Brumbaugh 48 Revised Summer 2016 3. Which of the three solutions was hypotonic to the Elodea canadensis cells? Explain your answer. 4. Which of the three solutions is isotonic to the Elodea canadensis cells? Explain your answer. 5. Would you expect pond water to be isotonic, hypotonic, or hypertonic to Elodea canadensis cells and why? Additional Questions: The external membrane of fish is semi-permeable. As is true for all animals, the materials that can pass freely without the aid of intra-membrane transport proteins are oxygen, carbon dioxide, and water. Any charged molecule such as Na+, Cl-, and any large molecules, such as sugars, requires the aid of transport proteins. All animals have salt (Na+, Cl-) in their blood. Answer the following questions on a separate sheet of paper. 1. What transport action could spontaneously occur in the external membrane of freshwater fish? 2. Now consider saltwater fish. What transport action could spontaneously occur in the external membrane of saltwater fish? Only consider the situation where the salt concentration is higher in the surrounding water than in the fish. 3. Using your text, or some other means, define both positive and negative feedback. Our bodies have the ability to sense potentially dangerous changes in blood pressure. When our blood pressure drops, our pituitary gland releases a hormone which enhances the ability of our kidneys to reabsorb water, thus making our urine more concentrated. Is this positive or negative feedback? Biology& 100 Laboratory Manual Mr. Brumbaugh 49 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 50 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: . Transporting Across Boundaries Exercise Name: . Use the diagrams below to determine the concentration gradients and expected direction of net flow for all the different molecules in each set-up. Determine the concentration gradient for each molecule type. Then draw an arrow showing the “expected” net movement of each molecule in the set-up. In each of these examples NaCl is permeable, while sucrose is impermeable to the membrane. A 100% H2O 35% NaCl 65% H2O B C 100% H2O 70% H2O, 10% sucrose, 20% NaCl 40% sucrose 60% H2O 60% NaCl 35% H2O 5% sucrose 1. Describe the net movements of the molecules in Condition A above. 2. Describe the net movement of the molecules in Condition B above. 3. Describe the net movement of the molecules in Condition C above. 4. Write out a hypothesis for each demonstration performed in this exercise on pages 40. Make sure your hypothesis is in the correct format! Biology& 100 Laboratory Manual Mr. Brumbaugh 51 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 52 Revised Summer 2016 Laboratory 4: Pea Lab: Scientific Method Applied Pre-lab Assignment Before coming to lab carefully read the previous pages on the Principles of the Scientific Method (Lab 1) and the following pages of Pea Lab: Scientific Method Applied then answer the pre-lab questions, page 59. Be prepared to hand in your responses to the pre-lab questions at the start of lab. Perspectives This lab is an opportunity to enhance your understanding and appreciation of the scientific method process in a semi-structured situation similar to that used by researchers in their work. Teams of students should carry out the activities in this lab. The division of labor is the responsibility of the team. The success of the group depends on the careful and conscientious effort of each person. This dependence on others is also characteristic of research and many other aspects of life (as you may already know). The work required for this lab spans two to four weeks depending on a number of factors. Your instructor will explain the methods of storage for your experimental set-ups and how to arrange for the use of the rooms and greenhouse to do your work. Exercise: Applying the Scientific Method Goals of this Lab Exercise To understand the mechanisms used in the scientific method Design an experiment and carry out the steps of a scientific experiment To work cooperatively in establishing a protocol for a scientific experiment Introduction In its simplest form, an experiment involves a check or control group compared with an experimental or test group. The control is held under constant conditions while the test group is exposed to the affects of various factors, one at a time. Any changes (dependent variable) that occur in the test group, but not in the control group, are assumed to be the result of the condition that is changed (independent variable). Each treatment, including the control, should be replicated, and the replicate organisms should be carefully distributed so that no individuals being treated will be favored more than others (control variables). In the activity that follows, you will investigate a small portion of a problem in biology that lends itself very neatly to the experimental method. It is concerned with coordination of growth and development in plants by chemical regulators called hormones. A disease of rice plants results in overly rapid growth of seedlings. The seedlings become tall and weak and finally fall over. Scientists found that a fungus caused the disease. Japanese scientists were able to produce symptoms of the disease with cell-free extracts of the fungus. From the extracts they isolated a substance, named gibberellin, which was shown to be the active agent causing the disease. Later research revealed that gibberellins are produced naturally by plants and are involved with regulating stem growth and other processes. In this project you will study the affect of gibberellic acid on pea plants whose genetic constitution (genotype) for the trait of height is dwarf. The expression of a genotype is termed its phenotype. The purpose of this lab is to determine whether the dwarf phenotype can be modified by the application of gibberellic acid to these plants. Biology& 100 Laboratory Manual Mr. Brumbaugh 53 Revised Summer 2016 Materials (per group of four students) 20 Little Marvel pea seeds and 2 flower pots Growth medium (vermiculite in greenhouse) Atomizer containing gibberellic acid solution Atomizer of de-ionized water & Safety Goggles Procedure Each team should decide on its organization, discuss the problem/hypothesis, and plan the experiment. Gather the materials needed and begin the activity. Prepare your seeds by following steps 1 through 6. 1. Seed Preparation - Place 20 pea seeds in a plastic cup and cover them with tap water so that the water level is about 2 cm. above the level of the pea seeds. Label the beaker with team identification and date. Place it in a dark cupboard in the biology lab and let the pea seeds soak overnight (process called imbibing) (i.e. 12-24 hours). The soaked seeds should now be planted as directed below. 2. After the seeds have soaked overnight take them to the greenhouse. Prepare two 15-cm flowerpots by adding a growth medium (vermiculite) to each pot. The containers should be about 3/4 full. Moisten the medium (might have to add more medium after watering) with tap water. In each pot, place ten soaked seeds and fill with more vermiculate to the brim of the pot, and moisten. Label each pot with team identification, date, and keep them in the greenhouse. Keep the medium moist, but not soggy wet. 3. When the seedlings are 2-3 cm. tall measure their height in millimeters. This is done by measuring the distance from the growth medium surface to the tip of the shoot apex. Measure all seedlings in the pot and average their heights. These lengths are the initial measurements or length zero. Wear Safety Goggles when spraying. Hormonal treatment of the plants follows immediately! Do the hormonal treatment outside of the greenhouse! 4. Using a hand atomizer (found in the refrigerator at the back of the lab room) containing gibberellic acid spray the plants of one pot as this will be your experimental group. Spray the other potted plants with the deionized water atomizer. This is the control group. Since some of the spray for the experimental treatment may drift, do all spraying outside of the greenhouse. Spray the plants until the leaves and shoot apex are wet enough to form droplets which will almost run off, but do not permit appreciable amounts to drop onto the growing medium. The spray treatment is done ONLY one time. Label each pot as “control” or “experimental.” Be sure that both of the pots are exposed to similar light conditions. Keep the growth medium of both pots uniformly moist, but do not spray water over the plants themselves. Keep both pots in the greenhouse. 5. Measure the average pea plant growth in each pot (not counting weekends) for five days and subtract the initial average from the time you first spray with the gibberellic acid and deionized water. When you measure record the heights of the plants in each pot, take note of the general health (leaf/stem color, leaf size, and stem diameter) and appearance of the plants, and record the data on Table 1 (page 57). In addition, on the last day, measure an inter-node length (Figure 1, page 56) on each plant and calculate the average internode length, and count the number of leaves produced on each plant in both the experimental and control groups. As the plants grow tall, it may be necessary to place stakes in the pots and tie the plants loosely to them. 6. At the conclusion of the activity, CLEAN UP all materials and equipment. Empty the used vermiculite into the appropriate container in the greenhouse. Biology& 100 Laboratory Manual Mr. Brumbaugh 54 Revised Summer 2016 Pea Lab Report: 1. Your research team will do a formal scientific paper write-up using the criteria established in Appendices A and C as your guide (abstract not included). Assign the different components (Title page, Introduction, Methods & Materials, Results, Discussion/Conclusions, and Reference Page) of the paper to various team members and turn in one report per team. 2. Answering the following questions will help your group to organize their thoughts for the scientific paper. The major question asked in this lab is “Can the phenotype of a genetically dwarf pea plant be altered by the addition of a plant hormone called gibberellic acid?” 1. Write a hypothesis using the “If .... Then” format for this experiment somewhere in the introduction. 2. On the last day of observations calculate and list the average inter-node lengths of the experimental and control plants. Include units of measure. Control average inter-node lengths = _________________ Experimental average inter-node lengths = ____________ 3. On your last day of observation calculate the average number of leaves in the experimental and control groups and list them below. Control average number of leaves = ________________ Experimental average number of leaves = ____________ 4. Prepare graphs of the average daily heights, number of leaves, node lengths, and any other relevant data for both the experimental and control groups. Properly title and label your graph (Appendix A). Place average height on the vertical axis and time on the horizontal axis. Graph both sets of data on the same graph by using different colors and a key. 5. What correlation(s) did you observe between number of leaves, inter-node length, and plant height? 6. Explain at least three possible sources of error that may have influenced the data you collected. 7. Suggest one additional experiment that would provide more valid data or show other pertinent results. Be specific!!! 8. Did you confirm your hypothesis? Explain using your data? 9. Can the phenotype of a genotypically dwarf pea plant be changed? Explain and support your answer by using specific numerical examples from the data collected. 10. According to this investigation which component of an organism’s life is more influential on its phenotype; its genetic make-up or the surrounding environmental influences? Explain using your data and/or observations to support your response. 11. If you were given the opportunity to apply gibberellic acid to your vegetable garden would you do so? Use your data and/or observations to support your response. 12. Does your data agree or disagree with previously published data (literature review Appendix F). Biology& 100 Laboratory Manual Mr. Brumbaugh 55 Revised Summer 2016 Figure 1 Plant Anatomy This drawing is to be used to guide the measurement of the inter-node length. Biology& 100 Laboratory Manual Mr. Brumbaugh 56 Revised Summer 2016 Data: Control Date: Experimental Initial height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Date: Height (mm.) Health of plants Inter-node Length (mm.) # of leaves Table 1 Pea Lab Data Table Use this table to record you raw measurement data and a description of the health of the plants. Miscellaneous, Observations, Information, and Notes Biology& 100 Laboratory Manual Mr. Brumbaugh 57 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 58 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: . Pea Lab: Scientific Method Applied Exercise Name: . Before coming to lab carefully read the previous pages on the Pea Lab: Scientific Method Applied then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. Note: Answer the following six questions before coming to lab, but after having read the previous pages of this handout! 1. What is gibberellic acid? 2. Define the following: phenotype- genotype- 3. Write a hypothesis using the "If .... Then" format for this experiment. 4. What is the independent variable of the pea experiment? 5. What is the dependent variable of the pea experiment? 6. Name at least three variables that you will be controlling in the pea experiment? Biology& 100 Laboratory Manual Mr. Brumbaugh 59 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 60 Revised Summer 2016 Laboratory 5: Energy Harvest – Fermentation in Yeast Pre-lab Assignment Before coming to lab carefully read the following pages of Energy Harvest – Fermentation in Yeast then answer the pre-lab questions, page 71. Be prepared to hand in your responses to the prelab questions at the start of lab. Perspectives In this lab activity you will determine which sugars (monosaccharide versus disaccharide) are best metabolized by yeast under anaerobic conditions and then propose hypotheses to explain why some sugars are metabolized but not others. Cultures around the world have for millennia used yeast fermentation to produce bread and alcoholic beverages. Yeast are able to metabolize some foods, but not others. In order for an organism to make use of a potential source of food, it must be capable of transporting the food into its cells and have the proper enzymes capable of breaking the food’s chemical bonds in a useful way. Sugars are vital to all living organisms. Yeasts are capable of using some, but not all sugars as a food source. Yeast can metabolize sugar in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen. When yeast respire glucose aerobically, oxygen gas is consumed at the same rate that CO2 gas is produced—there would be no change in the gas pressure in a test tube, but since they only produce CO2 gas during anaerobic respiration the CO2 gas pressure can be measured as a correlation to metabolism. The net equation for the more than two dozen steps involved in the aerobic respiration of glucose is: Enzymes C6Hl2O6(aq) + 6 O2(g) 6 H2O(l) + 6 CO2(g) + energy (36-38 ATP) glucose oxygen water carbon dioxide Although the aerobic fermentation of sugars is energetically much more efficient, in this experiment we will set the conditions so that the yeast can only complete the reactions anaerobically. When yeast ferment the sugars anaerobically CO2 production will cause a change in the pressure of a closed test tube system, since no oxygen is being consumed. We can use this pressure change to monitor the rate of respiration and metabolic activity of the organism. A gas pressure sensor will be used to monitor the fermentation of sugar. The alcoholic fermentation of glucose is described by the following net equation: Enzymes C6Hl2O6(aq) 2 CH3CH2OH(aq) + 2 CO2(g) + energy (2 ATP) glucose ethanol carbon dioxide Both anaerobic fermentation and aerobic respiration are multi-step processes that involve the transfer of energy stored in the chemical bonds of glucose to bonds in Adenosine TriphosPhate, ATP. The energy stored in ATP can then be used to perform cellular work: provide energy for biosynthetic reactions (e.g. growth and repair processes), move objects across membranes, etc. All organisms (i.e. monerans, protists, fungi, plants, and animals) utilize aerobic respiration and/or fermentation (anaerobic respiration) to produce ATP to power their cellular processes. Note that ethanol is a by-product of alcoholic fermentation (Figure 1). Ethanol, a 2-carbon alcohol, is also known as ethyl alcohol and, less correctly, simply as “alcohol”. Since yeast do not have the enzymes needed to metabolize ethanol, much of the energy stored in the molecules of glucose is trapped in the molecules of ethanol and is unavailable for use by yeast cells. The complete Biology& 100 Laboratory Manual Mr. Brumbaugh 61 Revised Summer 2016 breakdown of glucose to carbon dioxide and water in aerobic respiration yields much more energy than alcoholic fermentation: 36-38 ATP, versus only 2 ATP molecules produced by anaerobic respiration. Ethanol molecules produced by alcoholic fermentation diffuse from yeast cells into the surrounding aqueous environment. Since ethanol is harmful to cellular membranes yeast cells will die if ethanol concentrations reach a critical level, usually a concentration of about 12%. Figure 1 Glucose Summary Reactions This is a summary of three of the many possible fates of the 6-carbon sugar glucose under anaerobic and aerobic conditions. When anaerobic respiration occurs in animals (Figure 1) it is known as lactic acid fermentation since lactic acid, a 3-carbon organic acid, is the end product. Like alcoholic fermentation, lactic acid fermentation produces only 2 ATP. Perhaps if ethanol were produced anaerobically in animals more people would take up anaerobic sports such as sprinting or weight lifting! Since lactic acid is toxic to cells, anaerobic respiration can only occur for short periods of time in animals. In the presence of oxygen each lactic acid can be broken down to carbon dioxide and water. Aerobic respiration (Figure 2) occurs in three stages: glycolysis (involves soluble enzymes in the cytoplasm), Kreb’s cycle (uses soluble enzymes in the matrix of mitochondria), and the electron transport chain (a chain of reduction/oxidation paired electron carrier proteins called cytochromes found embedded into the inner membrane of the mitochondria). Alcoholic and lactic acid fermentation involve only glycolysis. Since both the Kreb’s cycle and the electron transport chain require oxygen to function, neither process can occur under anaerobic conditions. Glucose Plasma Membrane Glucose + NAD Cytoplasm Glycolysis NADH ATP Without O2 Present Ethanol + CO2 or Lactic Acid Pyruvate Mitochondrion O2 Present (Not drawn to scale!!) O2 Present Kreb’s Cycle NADH FADH2 CO2 ATP Carrier Proteins of the Electron Transport Chain ATP O2 H2O Figure 2 Glucose Metabolism Pathways Aerobic cellular respiration consists of glycolysis, Kreb’s cycle, and the electron transport chain. Anaerobic respiration involves only glycolysis and regenerates NAD + by either reducing pyruvate to produce lactic acid (animals), or by decarboxylating pyruvate to produce acetaldehyde (not shown) and then reducing acetaldehyde to produce ethanol. Under aerobic conditions the NADH produced by glycolysis enters the mitochondria of the cell where it becomes oxidized to regenerate NAD + by donating electrons to the electron transport chain, which results in the production of nearly 90% of the 36-38 ATP molecules produced per glucose molecule metabolized aerobically. Biology& 100 Laboratory Manual Mr. Brumbaugh 62 Revised Summer 2016 Exercise: Anaerobic Respiration Goals of this Lab Exercise Describe alcoholic fermentation and aerobic respiration, noting the reactants and products, and the relative energy efficiency of each Use a biology gas pressure sensor to determine which sugars are best metabolized anaerobically by yeast Propose reasonable hypotheses to explain why yeast can metabolize some sugars but not others Introduction Today’s exercise will afford you the opportunity to measure the rate of fermentation activity by yeast using different sugars. The equipment we will use measures the CO2 that is produced by the fermentation process. The production volume over time will be monitored and graphed by a computer program ready for your interpretation and analysis. The critical steps involve your ability to maintain a tight and consistent seal during each “run” of the exercise. Without a tight seal the CO2 produced will leak from the equipment and your data will be skewed. As with any experiment, the collection of the data is the easy part, but a clear understanding of the principles involved pose the greatest challenge. Make sure you understand the introductory material. Materials Computer and Safety Goggles Go-Link Interface Vernier Gas Pressure Sensor Yeast suspension and Disposable Transfer pipettes Basting bulb, thermometer, test tube rack Dropper bottles of: 5.0 % of glucose and sucrose Stopper assembly fitted with tubing 10 ml. Graduated cylinder Water bath (set at 37oC) 1L beaker (for water bath) 2 - 18 X 150 mm. test tubes Dropper bottles of: 5.0 % of lactose and fructose Procedure The Set Up 1. Prepare the computer for data collection by opening the Biology with Computers software as follows: Plug the CO2 gas sensor into the Go-Link connector. Go to Start click on Programs open Vernier open Logger Pro 3.5 open File open Biology with Vernier open “Exp. O6” Enzyme Pressure. The vertical axis has pressure scaled from ~90 to ~130kPa. The horizontal axis has time scaled from 0 to 15 minutes. 2. Adjusting the Valves to the Pressure Sensor. Open the valve (in the vertical position) on the rubber stopper assembly so that it is open to the atmosphere (Figure 3A). The closed position, used when monitoring the CO2 generated by the yeast, would have the knob in a horizontal position (Figure 3B). Figure 3A Open to the atmosphere Biology& 100 Laboratory Manual Mr. Brumbaugh Figure 3B Closed to the atmosphere 63 Revised Summer 2016 3. Prepare a water bath (Figure 4) at your table for the yeast to ensure that the yeast will remain at a constant and controlled temperature (Why?) when collecting data under data collection below. To prepare the water bath combine warm and cool water in a 1-liter beaker until it reaches 38 – 39oC. Fill the beaker with water until the beaker is ¾ full, but won’t spill over when the test tube containing the yeast and sugar is placed in it. Make sure to keep the water temperature constant at about 37oC. Figure 4 Experimental Set-up Experimental Set-up with reaction vessel in a water bath maintained at a constant temperature. 4. Obtain two large test tubes and label them 1 and 2. 5. Wear Safety Goggles when transferring solutions. Use a graduated cylinder to put 2.5 ml of one of your assigned sugars into test tube 1. You will do two “runs” for each of your groups assigned sugars and gather data from other groups to complete Table 1 (page 67) on the Report Sheet 6. Wear Safety Goggles when transferring solutions. Obtain the yeast suspension from the water bath at the back of the room. Constantly and Gently swirl the yeast suspension to mix the yeast that has settled to the bottom. Use a disposable transfer pipette to put 2.5 ml of yeast into a graduated cylinder and then into Test Tube 1. 7. Constantly and Gently swirl the yeast suspension while incubating for 10 minutes in the water bath set to 37oC at your table with the rubber stopper of the pressure sensor assembly firmly into Test Tube 1 and open to the atmosphere (Figure 3A) while Constantly and Gently swirling the yeast suspension in your water bath. Check that all connections are tight. Be sure to keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove about as much water as you will be adding or the beaker may overflow. Use a basting bulb to remove excess water. Data Collection 1. After the incubation period close the system to the atmosphere (Figure 3B). 2. Begin collecting data by clicking the green COLLECT button. Important: Constantly and Gently swirl the test tube while collecting data (this helps to liberate the carbon dioxide gas from the solution and helps to keep the contents mixed well). Monitor the temperature of the water bath. Be sure that it does not change by more than one degree. 3. Collect data until you are certain that there is a linear relationship between the pressure and time. Depending on the activity of the yeast, this usually takes 1 to 3 minutes. If the pressure exceeds 130 kPa, stop the computer by clicking the red STOP button. Open the air valve on the pressure sensor (Figure 3A) to prevent it from popping off! Data Analysis 1. Determination of the mean rate of fermentation. a. Move the cursor to the point where the pressure values begin to have a linear relationship. Hold down the mouse button, drag the cursor to the end of the linear section of the curve, and release the mouse button. Biology& 100 Laboratory Manual Mr. Brumbaugh 64 Revised Summer 2016 b. Click the “Analysis” button and then the “Statistics” button. A floating box will appear on the screen with the mean found in the box. Record this number in Table 1 of the Report Sheet (page 67) for the sugar and your group number. 2. Repeat using the same sugar and then perform two “runs” of your other assigned sugar. 3. Share your group’s data with the class and gather their data. Report 1. Turn in a group Report Sheet packet next lab by answering the Report Sheet questions on pages 67 through 69 as a group. Biology& 100 Laboratory Manual Mr. Brumbaugh 65 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 66 Revised Summer 2016 Report Sheet Lab Section: . Energy Harvest – Fermentation in Yeast Exercise Group Names: . . . . Data: Group No. Sugar Used Glucose Glucose Glucose Glucose Glucose Glucose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Lactose Lactose Lactose Lactose Lactose Lactose Fructose Fructose Fructose Fructose Fructose Fructose Rate of Respiration mean Table 1 Fermentation Exercise Data collected from the class. Rate of Respiration Average per sugar This table shows the fermentation rates of various sugars by yeast Analysis and Questions of Results: 1. Graph (your Figure 1) the class data on a separate piece of graph paper to show the average rate of respiration vs. sugar type. Label the graph fully and give it a proper title (Appendix A). Use the space below to interpret the trends seen on your graph by referring to the graph by its figure number. Biology& 100 Laboratory Manual Mr. Brumbaugh 67 Revised Summer 2016 2. Considering the results of this experiment, can yeast utilize all of the sugars equally well? Quote specific numerical values by citing your figure to answer this question. 3. Hypothesize why some sugars were not metabolized while other sugars were metabolized? 4. Hypothesize why the sugars that were metabolized were metabolized at different rates? Biology& 100 Laboratory Manual Mr. Brumbaugh 68 Revised Summer 2016 Refer to the perspectives of this lab to answer the following questions. 1. Write the overall balanced chemical equation for both aerobic respiration and anaerobic respiration of glucose by yeast and explain how they differ in terms of products. 2. Explain why there are different numbers of ATP produced when yeast metabolize glucose aerobically vs. anaerobically. Biology& 100 Laboratory Manual Mr. Brumbaugh 69 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 70 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: Energy Harvest – Fermentation in Yeast Exercise Name: . . Before coming to lab carefully read the previous pages on Energy Harvest – Fermentation in Yeast then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. 1. Define the following terms: aerobic respiration and anaerobic respiration? 2. Outline the steps or processes involved in the two types of respiration you defined in question 1 (see Figure 2). 3. Using your textbook or other sources, explain how enzymes are of such critical importance to the processes involved in respiration. 4. Speculate as to why yeast ferment pyruvic acid (make alcohol) differently from animals (make lactic acid). Biology& 100 Laboratory Manual Mr. Brumbaugh 71 Revised Summer 2016 Page to be used for Biological Doodling 6 Biology& 100 Laboratory Manual Mr. Brumbaugh 72 Revised Summer 2016 Laboratory 6: Mitosis and Online Karyotyping Pre-lab Assignment Before coming to lab carefully read the pages on Mitosis and Online Karyotyping and then answer the pre-lab questions, page 79. Perspectives In the late 1800’s new techniques to visualize cellular structure exploded with the discovery of vital stains and dyes. Most plant cells were fairly easy to visualize since most contain pigment molecules for photosynthesis, but animal cells were another matter. When viewed under a microscope lens sub-cellular structures, called organelles, were seen as simply the grainy consistency of the cytoplasm. When scientists would apply different pigments harvested from different plants the graininess took on distinct forms. Organelles could now be distinguished and studied as separate structures. Differences in cellular function could be attributed to the number and types of organelles found in various cells. By the end on the century scientist were using not only pigments from plants to stain cells, but were beginning to use heavy metals linked to pigments to further delineate structure. Dyes and stains became almost as an important discovery as the light microscope. The new technology of stains allowed a number of scientists to peer into cells like they could not have done before their discovery. One scientist, Walter Fleming, noted in salamander ovary cells that dark staining condensations appeared within the nucleus. These condensations were then separated toward opposite poles of the cell (“Dance of the Bodies”) just prior to the cell splitting into two new cells. He eloquently described the continuous stages of a process called mitosis. Today’s technology in the field of genetics and how genes affect the phenotype of an individual started years ago (mid-1800’s) with the work of Gregor Mendel. His work with pea plants set the stage for how chromosomes are sorted and passed from generation to generation. In the early part of the 1900’s another geneticist, Thomas Morgan, working with fruit flies (Drosophila melangastor) developed a technique called karyotyping that allowed him to visualize the structure of chromosomes. This technique harvested chromosomes from cells arrested between prophase and metaphase or pro-metaphase of mitosis, after being treated with colchicine to disrupt cellular membranes, the condensed chromosomes are then stained with a vital stain called giemsa, photographed/enlarged, and matched based on staining patterns and size. Harvesting the chromosomes at this time hopefully ensures complete condensation of the genetic material into individual units. This technique of karyotyping is used today to show the potential for genetic abnormalities within the genome of an individual. The specificity of the technique has been refined through the use of more specific stains (spectral analysis) which adhere to specific sites within the DNA molecules to further highlight the differences between chromosomes. Although mitosis is the most important process for ensuring that each daughter cell receives the correct amount of chromosomal material, mitosis is simply a portion of the life span of a cell. Following a nuclear division the cytoplasm is separated by a process called cytokinesis. In this process the animal parental cell aligns proteins called actin along the equator in animal cells and these proteins contract to pinch the membrane together forming a cleavage furrow along the metaphase plate to separate the daughter cells. In plant parental cells vacuoles containing cell wall material are lined across this plate. These eventually coalesce together to form a cell plate (eventually a cell wall) between the daughter cells (I wonder why we have always called them daughters and not sons?). Once cytokinesis is completed the cells move into the remaining portions of what is called the cell cycle. The cell cycle (Figure 1) is divided into interphase and division (either mitosis or meiosis followed by cytokinesis) or the life span of an individual cell. Interphase is further subdivided into the G1 period Biology& 100 Laboratory Manual Mr. Brumbaugh 73 Revised Summer 2016 (normal cell growth and function), the S period (DNA duplication), and the G2 period (synthesis of proteins, like actin, tubulin, and histone, involved with mitosis and cytokinesis). During interphase the normal day-to-day activities of the cell are carried out and the cell is said to be functioning normally. Figure 1 Cell Cycle The cell cycle of eukaryotic cells. http://staff.jccc.net/pdecell/celldivision/images/cellcycle.gif Exercise: On-line Karyotyping and Mitosis Goals of this Lab Exercise To understand the mechanisms of the cellular process called mitosis To understand the process and application of the technique called karyotyping To apply this knowledge to issues in today’s society in relationship to karyotyping Introduction In the following laboratory and on-line and activity you will draw and differentiate the phases of mitosis in botha plant and animal cells and play the role of a cytogenetic technician and complete the karyotype for three patients, then use these karyotypes to evaluate and diagnose each patient. Be careful! The emotional and physical well being of each patient is in your hands……or almost in your hands! Biology& 100 Laboratory Manual Mr. Brumbaugh 74 Revised Summer 2016 Materials (per group of four students) Compound light microscope Slides of onion root tips (Allium) Slides of white fish blastula PC computer with internet access Procedure Part 1: Visualizing the mitotic phases 1. Set-up two light microscopes at your lab table. 2. Have one group member obtain a prepared white fish blastula slide and another member a prepared onion root tip slide. 3. Under high power (40x), draw and label the different stages of mitosis (interphase, prophase, metaphase, anaphase, and telophase) for a plant and animal cell. Label the cells with terms such as centrioles, spindle tubules, sister chromatids, centromere, cell membrane, and cell wall. Make sure you attach the magnification and a drawing scale to your drawings. 4. With the assistance of your lab mates make a table comparing animal with plant mitosis and at the bottom of the table identify two differences in the strategies between animal and plant mitosis and cytokinesis strategies. Turn in your drawings next week with your Report Sheet. Part 2: On-line karyotyping 1. 2. 3. 4. Go to the Biology Project at: http://www.biology.arizona.edu/ Scroll down and click on “Human Biology”. Scroll down to “Activities” and then click on “Web Karyotyping”. Read the introduction and then complete the assignment as described. Record your responses on Table 1 page 77 of the Report Sheet. 5. Go to: http://www.scirus.com or do a search for karyotyping. 6. Search for a karyotyping website and answer the questions of page 77. Part 3: On-line Onion Root Tips: Phases of the Cell Cycle This activity is a digital version of a classic microscope lab. You will classify cells from the tip of an onion root into the appropriate phases of mitosis and then count up the cells found in each phase. You can use those numbers to predict how much time a dividing cell spends in each phase. In the process of doing this you will become familiar with the cell cycle and the process of mitosis and its stages, which are, oddly enough, the major goals of this activity! 1. Go back to the Biology Project at: http://www.biology.arizona.edu/ 2. Scroll down and click on “Cell Biology”. 3. Scroll down to “Activities” and then click on “On-line Onion Root Tips: Phases of the Cell Cycle”. 4. Read the introductory pages (about 3 total) and then complete the assignment as described. Record your responses in Table 2 of the Report Sheet page 77. Part 4: New Methods in Karyotyping: The Spectral Karyotype In this activity you will learn about a new technique for diagnosing chromosomal abnormalities, spectral karyotyping”. This technique is exciting because of its many applications, but also full of many controversial societal issues. On your report sheet are three questions pertaining to the old and new methods of karyotyping. Answer these questions on the report sheet as you do the following on-line reading assignment. 1. Go back to the Biology Project at: http://www.biology.arizona.edu/ 2. Scroll down and click on “Human Biology”. 3. Scroll down to “Activities” and then click on “New Methods in Karyotyping” Biology& 100 Laboratory Manual Mr. Brumbaugh 75 Revised Summer 2016 4. Read the introduction. 5. To learn about the methods involved click on “Methods” at the bottom of the “Introduction” page. 6. To learn about some of the possible applications of this new method click on “Applications” at the bottom of the “Methods” page. 7. Don’t forget to answer questions 1-3 located in the Report Sheet page 78 and turn in as a group. Biology& 100 Laboratory Manual Mr. Brumbaugh 76 Revised Summer 2016 Report Sheet Lab Section: . Mitosis and Online Karyotyping Exercise Group Names: . . . . Part 2: Web Karyotyping Data Patient Notation Diagnosis A B C Table 1 Web Karyotyping Data Information in this table shows the results of a web karyotyping exercise. Internet Search URL of Site: http:// . Title of Site: . Describe an interesting idea you learned at this site: Part 3: On-line Onion Root Tips: Phases of the Cell Cycle Interphase Prophase Metaphase Anaphase Telophase Total Number of Cells 36 Percent of Cells 100% Table 2 Mitotic and Cell Cycle Data Information in this table shows the results of an On-line Onion Root Tips: Phases of the Cell Cycle. What can be concluded about cellular tasks performed during each phase from the data collected above as it relates to cells in the cell cycle? Biology& 100 Laboratory Manual Mr. Brumbaugh 77 Revised Summer 2016 Part 4: New Methods in Karyotyping: The Spectral Karyotype 1. Explain how each of the following karyotyping methods work. The “old” method, Giemsa Dye Karyotyping: The “new” method, Spectral Karyotyping using fluorescent dyes: 2. List and then in your own words briefly discuss at least four possible applications of spectral karyotyping. 3. Identify and very briefly describe at least three controversial societal issues associated with spectral karyotyping. You will need to do some thinking here since the Biology Project website does not discuss any of the many issues involved. Biology& 100 Laboratory Manual Mr. Brumbaugh 78 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: . Mitosis and Online Karyotyping Exercise Name: . Before coming to lab carefully read the previous pages on Mitosis and Online Karyotyping then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. 1. Define the following terms: centriole, spindle tubule, sister chromatids, centromere, cell membrane, and cell wall. Draw a cell showing the location of these structures. 2. Using your text, outline the steps or phases of mitosis by describing the major events of each phase of the process. 3. Cite three reasons why cells would undergo mitosis and explain how mitosis fits into the cell cycle. 4. Cite when chromosomes are harvested for karyotyping during mitosis and explain why? Biology& 100 Laboratory Manual Mr. Brumbaugh 79 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 80 Revised Summer 2016 Laboratory 7: Mendelian Genetics Pre-lab Assignment Before coming to lab carefully read the pages on Mendelian Genetics then define the words at the end of the introduction section, page 82, on a separate sheet of paper. Perspectives In 1866 an Austrian monk, Gregor Mendel, presented the results of painstaking experiments on the inheritance patterns of garden peas. Those results were heard, but probably not clearly understood, by Mendel’s audience. Now, more than a century later, Mendel’s work seems elementary to modern–day geneticists, but its importance cannot be over stated. The principles generated by Mendel’s pioneering experimentation are the foundation for genetic counseling so important today to families with health disorders having a genetic basis. It’s also the framework for the modern research that is making inroads in treating diseases previously believed to be incurable. In this era of genetic engineering the incorporation of foreign DNA into chromosomes of unrelated species—it easy to lose sight of the basics of the process that makes it all possible. Geneticists depict an individual’s genetic make–up (or genotype) in a variety of different ways depending on the particular set of alleles they are working with. This may be unfortunate for the casual observer or the novice, but there are some commonalties that help to diffuse potential obfuscations. A gene would be the code for the expression of a certain trait say nose shape and for instance at a particular locus (site) on a chromosome you have an allele (an alternative expression of a gene received from a parent) for say a long nose and another allele on the paired chromosome of short or button nose shape. Individuals then can be classified as being homozygous (both alleles for a gene are the same) or heterozygous (both alleles are different for that trait). The most common system for identifying and relating genetic make–up is the use of capital and lower case letters. For instance at a particular locus (site) you have an allele (an alternative expression of a gene received from a parent). The dominant allele (a characteristic seen with an increased frequency in a defined population) would be expressed by a capital letter, say “B” and the recessive allele (a characteristic seen with a decreased frequency in a defined population and masked by the dominant allele) by the lower case letter “b”. Homozygous would mean that both alleles are the same and would be denoted by the same letter, whereas heterozygous would mean the alleles are different and be denoted by an upper and a lower case letter. Homozygous dominant individuals would be indicated by the notation BB and homozygous recessive individuals by the notation bb. Heterozygous individuals, those that have one dominant allele and one recessive allele, would be indicated by the notation Bb. This is called complete dominance. For the genetics of the ABO blood groups we use the capital letter “I” and then superscript the letter “i” with capital A’s, or B’s, or O’s to represent the alleles presence (IA, IB, IO). A similar type of notation occurs for other types of co-dominant genetic traits. Sometimes the apostrophe symbol is used to denote expression of a certain trait. Consider the following example of patterned baldness. Genotypes Men Phenotypes Women b´b´ Bald Bald b´b Bald Non-bald bb Non-bald Non-bald The apostrophe acts as an indicator of dominance in males and an indicator of recessives in women. What this actually denotes is the carrying of the trait for baldness on the b gene. Biology& 100 Laboratory Manual Mr. Brumbaugh 81 Revised Summer 2016 Another type of notation is the use of the symbols plus (+) and minus (-). The plus sign would indicate that the allele for the expression of a particular trait is present (usually the wild type or normal allele) and the minus sign would indicate that it is not present or that some mutated form of the normal gene is present. Sometimes we use the phrase wild type and symbol “wild” as a superscript to indicate the presence of the dominant naturally occurring allele. Often times you will see other sequences of letters that indicate the presence or absence of certain alleles. These short sequences are acronyms for a description of what the allele causes to be seen in the phenotype. In the case of simple dominance where a single dominant allele will mask the expression of a single recessive allele another nuance is added to the symbolic systems discussed. For example, a gene at a single locus controls tongue rolling. Individuals that can roll their tongues (phenotype) can have a genetic constitution (genotype) of either RR or Rr. Non tongue rollers have a genetic constitution of rr. If you observe a person who can roll his tongue, what is his/her genetic constitution? Well without looking at the parents maybe for more than one generation back and/or one or more generations of progeny the answer is either RR or Rr. This is because you receive half of your genes from each parent. So, if one is uncertain, how do you express the genetic constitution of these individuals? The answer is R ? . We know they can roll their tongue so we know that at least one of their alleles is the dominant R allele. Exercise: Mendelian Genetics Goals of this Lab Exercise To understand the mechanisms of Mendelian Genetics To understand the process and application of the technique used by geneticists Be able to apply this knowledge to pedigree and karyotyping analysis Introduction Through a series of activities we will examine some of the principles of genetics and techniques developed by geneticists to predict mating outcomes and understand how genetic information is passed from generation to generation. Before coming to lab, refer to your textbook or other references and write definitions for the following words: chromosome, genes, locus, allele, dominant allele, recessive allele, genotype, phenotype, gamete, haploid, diploid, monohybrid, dihybrid, homozygous, heterozygous, linked genes, autosomal chromosomes, sex chromosomes, sex-linkage, and homologous chromosomes. Materials Pages 83 through 93 of your lab manual Procedure 1. Pair up with a classmate. 2. Do each of the activities on pages and answer the questions in the space provided. 3. Turn in the completed Report Sheet (pages 83 through 93) as a pair. Biology& 100 Laboratory Manual Mr. Brumbaugh 82 Revised Summer 2016 Report Sheet Lab Section: . Mendelian Genetics Exercise Group Names: . . Activity A: Law of Segregation separating alleles into gametes: Tongue rolling (Able to form a Ushape when sticking out their tongue.) 1. What is the phenotype of an individual whose genotype is RR? 2. What is the phenotype of an individual whose genotype is Rr? 3. What is the phenotype of an individual whose genotype is rr? 4. What are your phenotype and genotype for tongue rolling? The distribution of alleles during the formation of gametes was one of the principles described by Gregor Mendel. It is called the Law of Segregation. The two alleles of a gene segregate, or separate, from each other so that each one ends up in a different gamete. 1. If a person’s genotype is RR, what are the genotypes of the resulting gametes? 2. If the person’s genotype is rr, what are the genotypes of the resulting gametes? 3. If the person’s genotype is Rr, what are the genotypes of the resulting gametes? 4. If your phenotype was tongue roller, what would you have to find out in order to know your genotype for sure? Activity B: Ear Lobes An unattached earlobe (F) is dominant to attached earlobes (f). We can only guess at the biological significance of the kind of earlobe might make. Did your grandmother or mother use yours a lot? 1. What is the phenotype of an individual whose genotype is FF? 2. What is the phenotype of an individual whose genotype is Ff? 3. What is the phenotype of an individual whose genotype is ff? Biology& 100 Laboratory Manual Mr. Brumbaugh 83 Revised Summer 2016 4. What is your phenotype and genotype with respect to earlobe attachment? 5. If a person’s genotype is FF, what are the genotypes of the resulting gametes? 6. If the person’s genotype is ff, what are the genotypes of the resulting gametes? 7. If the person’s genotype is Ff, what are the genotypes of the resulting gametes? 8. If your phenotype was unattached earlobe, what would you have to find out in order to know your genotype for sure? 9. Joe Buxtawhody and the members of his immediate family have attached earlobes. His maternal grandfather has unattached earlobes. What is the genotype of his maternal grandfather? 10. His maternal grandmother is no longer living. What could have been the genotype of his maternal grandmother? PTC (phenylthiocarbamide) is an anti-thyroid drug that prevents the thyroid gland from incorporating iodine into the thyroid hormone. The ability to taste PTC is associated with the functioning of the thyroid gland. As with many patterns of inheritance, the nature of the relationship between “tasting” and disease is unknown. The ability to taste PTC is an autosomal (found on one of the chromosomes other than the sex chromosomes) trait. Tasting (T) is dominant to non-tasting (t). 1. Can you taste PTC? 2. What is your genotype? 3. How do you know? Activity C: The chromosomal basis of independent assortment: a monohybrid model. When the genotypes of the parents are known, we may determine what gametes the parents can make and in what proportion the gametes will occur. This information allows us to predict the genotypes and phenotypes of the offspring. The prediction is simply a matter of listing all the possible combinations of gametes. In this section your will be doing monohybrid (one trait) crosses. By convention, the parental generations are called P. The first generation of offspring is called F1. F stands for filial, which refers to a son or daughter, so F1 is the first filial generation. If members of the F1 generation are crossed, their offspring are called the F2 generation and so on. Biology& 100 Laboratory Manual Mr. Brumbaugh 84 Revised Summer 2016 Predict the results of the following cross using T and t to denote tasting and non-tasting, respectively P generation TT x TT 1. What genotypes will be found in the F1 generation? 2. What phenotype(s) will be found in the F1 generation? 3. Explain why you made these predictions. Predict the results of the following cross: P generation TT x tt 1. What genotypes will be found in the F1 generation? 2. What phenotype(s) will be found in the F1 generation? 3. Explain why you made these predictions. The previous examples were fairly simple since the parents were only able to produce one type of gamete. However, the complexity escalates rapidly when parents can produce more than one type of gamete. To deal with the presence of more than one type of gamete we employ the Punnett square. This technique was developed by the geneticist Reginald Punnett in 1910 as a means of showing the probabilities of progeny outcomes. Consider the cross between the F1 progeny above, Tt, to produce the F2 generation. The F1 Cross is Tt x Tt 1. What type or kind of gametes can the first parent produce? 2. What type or kind of gametes can the second parent produce? T 3. The Punnett square would look like this. t T t Biology& 100 Laboratory Manual Mr. Brumbaugh 85 Revised Summer 2016 4. Fill in the Punnett square. 5. What are the possible genotypes in the F2 generation? 6. What are the phenotypes of each genotype in the F2 generation? 7. What is the genotypic ratio of this cross? 8. What is the phenotypic ratio of this cross? 9. Joe cannot taste PTC, but both his mother and his father can taste PTC. Do a Punnett square to calculate the expected phenotypic ratio among Joe’s siblings. Activity D: The chromosomal basis of independent assortment: a dihybrid model. Genes that are located on the same chromosome are linked with each other. If genes are located on separate, non-homologous chromosomes, they are not linked, or unlinked. Unlinked genes separate independently during meiosis (gamete formation). For example, consider the allelic pair T and t and a second allelic pair F and f. If the T gene and the F gene are not linked, their alleles can be found in any combination in the gametes. That is, the T allele can be in the same gamete as either the F allele or the f allele. This is Mendel’s Law of Independent Assortment. The word assortment in this case refers to the distribution, or sorting, of alleles into gametes. Assume the circle below represents a cell. Finish drawing the cell adhering to the following conditions: a diploid cell, with two homologous pairs of chromosomes, with two unlinked genes called T and F, and the cell is heterozygous with respect to both of these genes. 1. What is the genotype of this cell? 2. The T gene is for tasting PTC and the F gene is for unattached earlobes. What is the phenotype of the individual represented by this cell? 3. Recall that when this cell undergoes meiosis, each gamete receives one member of each homologous pair. List the possible combinations of alleles that will be found in the gametes. Biology& 100 Laboratory Manual Mr. Brumbaugh 86 Revised Summer 2016 4. In what proportion would you expect these gametes to occur? The resulting phenotypic ratios in the F2 generation of a dihybrid cross (2 traits) can be quite different than those observed from a monohybrid cross. But the process is essentially the same. First you list all possible gametes each parent and subsequent parents can produce. Second, you then assign the gamete possibilities to the Punnett square and fill it in. Finally you count the progeny and determine the number of progeny in each phenotypic category. Remember, when determining the types of gametes possible, each gamete must have one member of each homologous pair of chromosomes. For example, if you are considering a T gene (ability to taste PTC) and an F gene (unattached earlobe), each gamete must have one allele for the T gene (either T or t) and one allele for the F gene (either F or f). 1. What type of gametes will the following genotypes produce? Genotype: TTFF Gametes . Genotype: TtFF Gametes . Genotype: ttFf Gametes . Genotype: TtFf Gametes . Cross or mate a homozygous tasting and unattached earlobe parent with a homozygous nontasting attached earlobe parent? 2. What are the genotypes of these two parents? 3. What type of gametes can each parent produce? 4. How many different types of gametes can each parent produce? Record the genotypes of the gametes each parent can produce. 5. Construct the Punnet square that shows the cross between the parents. This can be tricky if you don’t understand gamete production from the parents. 6. What are the possible genotype(s) of the F1 progeny? 7. What are the phenotype(s) of the F1 progeny? 8. How many different types of gametes can the F1 parent produce? Record the genotypes of the gametes each parent can produce. Biology& 100 Laboratory Manual Mr. Brumbaugh 87 Revised Summer 2016 9. Construct the Punnet square to show the possible outcomes of a dihybrid cross between a mating of the F1 offspring. (Hint: Look at the figure on page 94 of the lab manual.) 10. What is the expected genotypic ratio of the F2 progeny? 11. What is the expected phenotypic ratio of the F2 progeny? 12. What would the expected phenotypic ratio of the F2 progeny be if the T gene and the F gene were linked? 13. A couple with the genotypes TtFf and TtFf have 16 children. Twelve of them can taste PTC and have unattached earlobes; the other 4 can’t taste PTC, 2 of the 4 have attached earlobes and 2 of the 4 have unattached earlobes. Why doesn’t this family match the expected ratio? (Hint: If the probability of having a male child is 50% why can one family have 7 daughters and no sons?) Biology& 100 Laboratory Manual Mr. Brumbaugh 88 Revised Summer 2016 Activity F: The ABO blood groups ABO blood groups are the most commonly known blood groups. Rh factor is another commonly known blood group. But in all fairness humans are much more complicated than that. There are currently over 300 different types of blood factors known to hematologists. ABO is an acronym for the three types of alleles an individual may potentially have. Of course a diploid individual can only have 2 alleles. The genotypes and phenotypes of different combinations of the three alleles are given in Table1. Phenotypes Phenotypes A B AB Genotypes Genotypes AA, AO or IA IA , IAi BB, BO or IB IB,, IBi AB or IA IB Table 1 Blood Types These are the most common blood type phenotypes and genotypes seen in humans. Notice that phenotypes A and B can have two possible genotypes. Notice also that blood types AB and O only have 1 possible type of genotypes. This situation is a pattern of inheritance referred to as co-dominance. The allele A is dominant to the allele O. The allele B is dominant to the allele O, but the allele A is co-dominant to the allele B, hence the phenotypic blood type AB. The Rh blood factor’s pattern of inheritance is the case of simple dominance that we have been assuming in this lab till now. The Rh blood factor is inherited as a single pair of alleles. Rh positive (Rh+) is dominant to Rh negative (Rh-). Answer the following questions. 1. A boy wonders if he is adopted. He compares his blood type to those of his parents. a. If the father is blood type AB and the mother is blood type O, what blood types would indicate that the child might have been adopted? b. If the father is blood type A and the mother is blood type B would blood typing help the boy determine if he was adopted? 2. If you are Rh+ can you know your genotype for sure? Why? 3. If you are Rh+ and everybody in your family is Rh+ (parents, siblings, offspring) what is/are your probable genotype? Why? Do you know your genotype with absolute certainty? Explain. Biology& 100 Laboratory Manual Mr. Brumbaugh 89 Revised Summer 2016 Activity G: Color Blindness Color blindness is a sex–linked recessive trait (Table 2). Sex linked means that the trait would be localized to one of the two sex chromosomes. The possible genotypes and phenotypes are given below. Females Genotypes B X XB Males Genotypes Phenotypes normal vision XBY Phenotypes normal vision XBXb normal vision XbY color blind XbXb color blind Table 2 Sex Linkage These are the most common designation for color blindness in humans. 1. Are you color blind? 2. If you are (or were) color blind, what is your genotype? 3. If you are a female and are not color blind, you can judge whether you are homozygous or heterozygous by knowing if any member of your family is color blind. a. If your father is color blind, what is your genotype? . b. If your mother is color blind what is your genotype? . c. If you know of no one in your family who is color blind, what is your probable genotype? . 4. The only member of Josephine’s family who is color blind is her brother. a. What is her brother’s genotype? . b. Her father’s genotype? . c. Her mother’s genotype? . d. What is Josephine’s genotype if she later has a color-blind son? . Activity H: Determine the genotype of the unborn Through genetic counseling, it is sometimes possible to identify parents who are likely to produce children with genetic disorders. And then it is sometimes possible to test fetal cells to determine if the newborn does indeed have the disorder. Pedigree charts can be constructed to show the inheritance of a genetic disorder within a family. Thereafter, it may be possible to determine whether any particular individual has an allele for that disorder. Then a Punnett square can be done to determine the chances of a couple producing an affected child. This process is called analysis by pedigree charts. Biology& 100 Laboratory Manual Mr. Brumbaugh 90 Revised Summer 2016 Some genetic disorders are discovered following amniocentesis, a procedure that allows a physician to withdraw a portion of the amniotic fluid and thereby fetal cells by means of a long needle. The fetal cells are cultured and then a karyotype of the chromosomes is prepared. A karyotype shows all the chromosomes of the individual arranged by homologous pairs (analysis by karyotyping, refer to Lab 8 for information about karyotyping). Homologous chromosomes have the same size and shape. Karyotypes can show genetic aberrations. For instance, in humans, if you have an extra chromosome 21 you will have Down syndrome. Geneticists can now map human chromosomes, that is, they can find the exact loci for various genes. If the exact locus for a mutant gene causing a genetic disorder is known, geneticists can make copies of the gene and use these copies to test the chromosomes for the disorder. This is called analysis by genetic markers and involves the use of DNA probes (the copies of mutant genes) and restriction enzymes that cleave the DNA into manageable sizes for analysis. Analysis by pedigree charts There are three types of inheritance patterns you need to be aware of to complete this portion of the activity: Autosomal dominant shows with an increased frequency in a defined population Autosomal recessive shows with a decreased frequency in a defined population Sex–linked recessive traits linked to the sex chromosomes A trait that is an autosomal (trait is associated with one of the chromosomes number 1 through 22) dominant trait only needs one copy of the allele for the individual to be affected. A trait that is an autosomal recessive trait will require two copies of the recessive trait to be present in order for the individual to be affected. Sex–linked recessive traits primarily affect men not that women are totally excluded but the likely–hood of a women being affected is lower (Why would this trend be true?). Look at the following table (Table 3) to see the possible genotypes and phenotypes for some common single gene inheritance patterns. Inheritance Pattern Autosomal Dominant Autosomal Recessive Sex–linked Recessive Genotype BB Bb bb BB Bb bb XB XB XB Xb XbXb XB Y XbY Phenotype Affected Affected Not affected Not affected Not affected Affected Normal female Normal female Affected female Normal male Affected male Table 3 Pedigree Analysis These are the most common designations for pedigree assignment in humans. Consider the following three pedigrees. Using Table 3, determine the pattern of inheritance and indicate the probable genotype for each individual in each pedigree (the analysis). See page 82 to assist with your genotypic identification of the unknown members of the pedigree. Biology& 100 Laboratory Manual Mr. Brumbaugh 91 Revised Summer 2016 Pedigree #1 I 1 Male 2 II Female 2 Unaffected Affected 1 2 3 III 1 Analysis: (Why did you label the genotypes of the family members the way you did?) Pedigree #2 I 1 2 3 4 II 1 2 3 4 5 III 1 Analysis: Biology& 100 Laboratory Manual Mr. Brumbaugh 92 Revised Summer 2016 Pedigree #3 I 1 2 3 4 II 1 2 3 4 5 III 1 Analysis: Biology& 100 Laboratory Manual Mr. Brumbaugh 93 Revised Summer 2016 Page to be used for Biological Doodling Biology& 100 Laboratory Manual Mr. Brumbaugh 94 Revised Summer 2016 Laboratory 8: Modeling DNA Structure, Replication, & Protein Synthesis Pre-lab Assignment Before coming to lab, carefully read the following pages on Modeling DNA Structure, Replication, & Protein Synthesis and then answer the pre-lab questions, pages 107 and 108. Be prepared to discuss and hand in your responses to the pre-lab questions at the start of lab. Perspectives This investigation differs from those you have completed up to this point. You will use various kinds of models to learn how DNA controls the activities of cells. Many scientists use models to understand biological processes. Watson and Crick used models to figure out the structure of DNA and scientists use models today to study biological problems, from the structure of proteins to making predictions concerning how environmental factors may influence entire ecosystems. In this investigation, you will work collaboratively with your partners to propose a structure for DNA, show how DNA acts as a template to make RNA, and how RNA is used as a template to make protein. To accomplish these tasks you will use models of the building blocks of DNA, RNA, and protein to represent DNA replication and protein synthesis. Though it comes as no surprise that the composition of DNA between different organisms is different, it is not immediately obvious why the muscle cells, blood cells, and brain cells of any one particular vertebrate are so different in their structure and composition when the DNA of every one of their cells is identical. This is the key to one of the most exciting areas of modern cell biology. In different cell types, different sets of the total number of genes (genome) are expressed. In other words, different regions of the DNA are "active" in the muscle cells, blood cells, and brain cells. Central Dogma of Biology: DNA RNA Protein (Product) Phenotype To understand how this difference in DNA activity can lead to differences in cell structure and composition, it is necessary to consider what is often known as the central dogma of molecular biology (Figure 1): "DNA copied into RNA and RNA is read into protein”. In molecular terms, a gene is that portion of DNA that encodes for a single protein. The dictum "one gene makes one protein" has required some modification with the discovery that some proteins are composed of several different polypeptide chains, but the "one gene makes one polypeptide" rule does hold. Figure 1 Central Dogma The central dogma of biology states that DNA contains a genetic code that allows it to make copies of itself. An essential group of proteins, called enzymes, act as biological catalysts and regulate all aspects of cell metabolism and conspire to complete the steps of DNA replication and protein synthesis or essentially they regulate you. Their role in DNA replication and protein synthesis are vital in maintaining the integrity of these molecules to ensure continued functioning of the cell. In fact the structure of the enzyme is encoded into the DNA molecule. Biology& 100 Laboratory Manual Mr. Brumbaugh 95 Revised Summer 2016 The Structure of DNA: Nucleotide strands DNA is a double helix, with about 10 nucleotide pairs per helical turn (Figures 2 and 3) in an antiparallel arrangement (5’ to 3’ and 3’ to 5’ based on the position of the carbon’s in the ring of the sugar (Figure 5)). Each spiral strand is composed of a phosphate group (P), a sugar (deoxyribose) (S), and an attached base (B) or P-S-B that is connected to a complementary strand by hydrogen bonding between paired bases, adenine (A) with thymine (T) and guanine (G) with cytosine (C). Two hydrogen bonds (weak non-covalent bonds) connect adenine and thymine, while guanine and cytosine are connected by three. James Watson and Francis Crick first described this structure in 1953. Figure 2 DNA Structure DNA is a double stranded molecule, each strand consisting of a chain of nucleotides. Each nucleotide consists of a phosphate group, a deoxyribose sugar, and a nitrogen containing base (Guanine Cytosine, Adenine, or Thymine). Weak hydrogen bonds between the bases of each strand hold the two strands together. Figure 3 DNA Structure An illustration of the double helical structure of the DNA molecule. Nucleic acids are long, chain-like molecules formed by the linking together of smaller molecules called nucleotides. The nucleic acid DNA or DeoxyriboNucleic Acid is the material from which genes are made. Watson and Crick used information gathered by other researchers to make models of DNA in 1953. Their models led them to make one of the greatest scientific discoveries of the last century, the determination of the structure of DNA. The sugar and the phosphate part of each DNA nucleotide are the same, but the bases differ. There are four different types of nitrogen bases in DNA nucleotides—thus there are four different types of DNA nucleotides. Biologists typically refer to the type of nucleotide by its first letter: A = adenine, C = cytosine, G = guanine, and T = thymine. Biology& 100 Laboratory Manual Mr. Brumbaugh 96 Revised Summer 2016 Notice the polarity of DNA and RNA. There is always a 3’ and 5’ prime end of each strand (Figure 4) due to the number sequence of the carbons in the nucleotide sugar (Figure 5). The phosphate is always attached to the 5’C of the nucleotide sugar (either deoxyribose or ribose) and the nucleotide base is always attached to the 1’C of the sugar. This orientation leaves the 3’C available to attach to the next nucleotide at its phosphate end (5’C). Why is this specific orientation required in both DNA and RNA? Figure 4 Representation of DNA Replication The two strands of the original DNA molecule separate, and then each serves as a template in the formation of two new DNA molecules that will have the identical base sequence as the one original DNA molecule. Figure 5 Sugar Carbon Ring Numbering Each carbon in a sugar ring is numbered starting with the carbon to the right of the oxygen in the ring. DNA Replication: Semi-conservative replication To reproduce (Why do cells reproduce?) a cell must first copy and transmit its genetic information (DNA) to all of its progeny. To do so, DNA is replicated (During what part of the cell cycle?) , following the process of semi-conservative replication (Figure 4) by laying down the new bases in a 5’ to 3’ direction by reading the original strand in a 3’ to 5’ direction. Because of this orientation the replication process follows one strand in a straight forward direction (leading strand) while the opposite strand is read in short sequences (called Okasaki fragments) in the opposite direction from the leading strand but in the correct direction in terms of 5’ to 3’ (lagging strand). Each strand of the original molecule acts as a template for the synthesis of a new complementary DNA molecule. In the last section of their paper, Watson and Crick added this statement: “It has not escaped our notice that the specific base pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”. The process they were thinking of involved the separation of the two strands of DNA by an enzyme known as DNA Helicase. Once open another enzyme called RNA Primase lays down a short sequence using RNA bases as a primer to direct the enzyme called DNA polymerase III to read the original strand and lay down the appropriate base in the opposite position to build a new strand of DNA. Another enzyme, called DNA polymerase I, comes along and reads the primers as RNA and subsequently removes this portion and adds the appropriate DNA bases to complete the new strand and finally DNA Ligase ties all the loose ends together such that two Biology& 100 Laboratory Manual Mr. Brumbaugh 97 Revised Summer 2016 single-stranded sections of DNA are formed from one double-stranded molecule or from replication. The key is the consistence to hopefully avoid copying issues or mutations. DNA RNA: Transcription RNA (RiboNucleic Acid) is produced using DNA as a pattern. During this process, called transcription, the genetic code is transferred from DNA to RNA. During transcription the two strands of a DNA molecule become separated by a group of enzymes known as transcriptions factors along part of the molecule’s length. One strand, the non-coding strand, remains inactive (maybe used at another time), but the other, the coding strand, is used as a template to synthesize RNA, a single stranded molecule. The enzyme responsible for transcription, RNA polymerase, like DNA polymerase (III or I), can only build RNA in the 5’ to 3’ direction (Why?). Therefore, begin transcription at the 3’ (sugar) end of the DNA molecule by pairing each DNA nucleotide with its RNA complement. The base pairing rules are the same as in DNA, except Uracil pairs with Adenine since RNA does not contain Thymine. Before the synthesis of a protein begins, the corresponding RNA molecules messenger (mRNA), transfer (tRNA), and ribosomal (rRNA) are produced by RNA transcription (Figures 5 and 6) by reading the DNA strand in a 3’ to 5’ direction and laying RNA bases down in 5’ to 3’ direction. One strand of the DNA double helix is used as a template (coding strand) by the enzyme RNA polymerase (under the direction of the transcription factors) to synthesize RNA. The RNA’s migrate from the nucleus to the cytoplasm. During this step, RNA’s go through different steps of maturation including splicing out non-coding sequences (called introns (these regions do not code for product as far as we now know)) from coding sequences (called exons (both sides of the DNA code for product in these regions)) and adding a GTP cap (not shown) to the 3’ end and a poly A tail (not shown) to the 5’ end of the molecule. The coding sequence for a particular amino acid of the mRNA can be described as units of three nucleotides called codons. Figure 5 DNA Transcription RNA polymerase faithfully copies DNA to produce RNA molecules. Figure 6 DNA Transcription During transcription the two strands of a DNA molecule become separated along part of the molecule’s length. Only one of the two strands of DNA, the coding strand (the bottom strand in this case) acts as a template during transcription. The enzyme RNA polymerase reads the coding strand to produce a single stranded RNA molecule by following the base pairing rules used in DNA, with one exception—since thymine is not found in RNA, uracil pairs with adenine. Take Note... Transcription produces three major types of RNA which all get transported from the nucleus through a nuclear pore to the cytoplasm of the cell. Ribosomal RNA (rRNA): Combines with proteins in the cytoplasm to form ribosomes, the protein making factories of the cell. Messenger RNA (mRNA): Brings the instructions for protein synthesis (the genetic code) from DNA in the nucleus to the ribosomes. Biology& 100 Laboratory Manual Mr. Brumbaugh 98 Revised Summer 2016 Transfer RNA (tRNA): Combine with amino acids in the cytoplasm and transport them to the ribosome where tRNA interacts with ribosomes and mRNA to link the amino acids together to form proteins. There is a different tRNA molecule for each of the 20 amino acids. Each tRNA molecule consists of about 75 nucleotides. At one end of each tRNA molecule is a three base sequence called the anticodon, which are complementary to one of the codons in mRNA. An activating enzyme called aminoacyl-tRNA synthetase can attach a specific amino acid to the opposite end of the tRNA molecule. This enzyme is specific for a particular amino acid and a particular tRNA: each tRNA can carry only one kind of the 20 naturally occurring amino acids. RNA Protein: Translation The process of translation begins with the binding of a ribosome to the mRNA at the start (or initiation) codon, AUG. The ribosome proceeds to slide down the RNA molecule reading its message three bases (i.e. one codon) at a time (Figure 7). During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the anticodon (Figure 8) of the transfer RNA (tRNA). The ribosome (containing a short strand of rRNA) moves from codon to codon along the mRNA as amino acids are added one by one, translated into polypeptide sequences dictated by DNA and represented by mRNA (Figure 7). At the end, a releasing factor binds to a stop codon, terminating translation and releasing the complete polypeptide (protein) from the ribosome. Since one specific amino acid can correspond to more than one codon, the genetic code is said to be redundant. Figure 7 RNA Translation or Protein Synthesis During protein synthesis, ribosomes move along the mRNA molecule and "read" its sequence three nucleotides at a time (codon) from the 5' end to the 3' end. Each amino acid is specified by the mRNA's codons. Each codon pairs with a specific anticodon, a sequence of three complementary nucleotides at one end of a tRNA molecule. Since each tRNA molecule carries a specific amino acid at one end, the order of codons on the mRNA molecule determines the order of amino acids to be linked together during protein synthesis. Proteins then are long chains of amino acids constructed by varied arrangements of the 20 different amino acids. As the proteins are released from the ribosome, they fold into unique shapes (conformation) that depend on the particular sequence of amino acids in the chain. Hence, it is the protein’s primary structure (i.e. the order of the amino acids in the protein), which is encoded in the gene and faithfully transcribed to produce mRNA, which in turn is translated by ribosome’s into an amino acid chain, that determines the three-dimensional structure of a protein, and thus its particular function. The human body possesses some 30,000 different kinds of proteins and several million copies of many of these. Each plays a specific role. For example, hemoglobin carries oxygen in the blood; actin and myosin interact to generate muscle movement, and acetylcholine receptor molecules mediate chemical transmission between certain nerve and muscle cells. The versatility of proteins, the workhorse molecules of the cell, stems from the immense variety of molecular shapes that can be created by linking amino acids together in different sequences. The Biology& 100 Laboratory Manual Mr. Brumbaugh 99 Revised Summer 2016 smaller proteins consist of only a few dozen amino acids, whereas the larger ones may contain in excess of 200 amino acids, all linked together in a linear chain by peptide bonds. There exact sequence dictated by DNA and represented by mRNA is constructed through the joint processes of transcription and translation. Translation of the mRNA molecule involves each type of RNA. The ribosome (40S and 60S template) is attached to the mRNA by reading a start codon. The shape of the ribosome allows for only two tRNA’s to match their individual anti-codons to respective codons within the ribosome at one time. These matching sites are called the ribosome P and A sites, respectively. Enzymes found outside the ribosome detach the amino acid bonded to the first amino acid and attach it the second tRNA’s amino acid. The ribosome is then shifted to open the next codon and the steps repeat to lengthen the building protein one amino acid at a time, until a stop codon (no tRNA matches the codon of the three stop codons) is read at the end of the mRNA. Once the stop codon is read the resulting protein is released (What happens to it to become functional?) from the last tRNA, the ribosome is removed, and the message is recycled. Figure 9 and Table 1 are based on the three codon bases of mRNA. The Genetic Code: Three Base Sequences The process of identifying the sequence of amino acids in a protein, then reading them back into mRNA codons, and then to DNA base sequences began in the 1930’s by work done by Tatum and Beadle. In 1961 Nirenberg proved that by repeatedly linking uracil (UUUUUUU) into an mRNA the resulting protein contained only one amino acid (phenylalanine). From this beginning molecular biologists have identified the amino acid that is associated with each of the mRNA codons. The following figures (Figure 8 and 9 plus Table 1) show this relationship and also identifies the special start and stop codons. Since RNA is constructed from four types of nucleotides, there are 43 or 64 possible triplet sequences or codons. One of these codons plays a dual role in mRNA. If AUG is read in the mRNA sequence it signifies placing a methionine in that position, but when the AUG is placed at the beginning of the mRNA it also indicates where the rRNA is attached to begin the process of translation or the start codon. Three other codons (UAA, UAG, or UGA) specify the termination of the polypeptide chain and are called the "stop codons" (What happens to each of the RNA’s when translation is completed?). The remaining 61 codons are used to specify the other 19 different amino acids. Therefore, most of the amino acids are represented by more than one codon and the genetic code is said to be redundant, except for UGG (codes for tryptophan) (Why?). Figure 8 Translation The Pairing of a Codon in mRNA with an Anticodon of the tRNA inside a ribosome with rRNA (not shown). Biology& 100 Laboratory Manual Mr. Brumbaugh 100 Revised Summer 2016 Figure 9 The Genetic Code The three bases of an mRNA codon are designated here as the first, second, and third bases, reading in the 5' to 3' direction along the mRNA. Note that UGG is the only codon for the amino acid tryptophan, but most amino acids are specified by two or more codons. For example, both UUU and UUC code for the amino acid Phenylalanine. When either of these codons is read by a ribosome moving along an mRNA molecule, Phenylalanine will be incorporated into the growing protein molecule. Think of UUU and UUC as synonyms in the genetic code. Note that AUG codes for the amino acid methionine but also functions as a “START” signal for ribosomes to begin translating the mRNA at that location. Three of the 64 codons function as "STOP" signals. Any one of these termination codons marks the end of the genetic message. Ala: Alanine Cys: Cysteine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Gln: Glutamine Glu: Glutamic acid Gly: Glycine His: Histidine Ile: Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Val: Valine Trp: Tryptophan Tyr: Tyrosine Table 1 Amino Acid Abbreviations This table shows the abbreviations for each of the twenty different amino acids that are used to build proteins coded for in the DNA Exercise: Modeling DNA Replication and Protein Synthesis Goals of this Lab Exercise Describe the components of DNA and RNA nucleotides Explain how DNA is replicated within a cell and use models to model the process Explain how DNA is transcribed to produce RNA and use models to model the process Explain how mRNA is translated into protein and describe the role of each of the following in the process: mRNA, tRNA molecules, amino acids, and ribosomes Determine the amino acid sequence of a protein when given the base order of the coding or non-coding strand of a gene Compare and contrast the possible effects each of the following point mutations have on the amino acid sequence of a protein: a single base substitution, a single base deletion, and a single base addition Biology& 100 Laboratory Manual Mr. Brumbaugh 101 Revised Summer 2016 Introduction This lab is actually divided into two activities (building a model and answering questions) that are reliant on each other. For you to complete each of the activities a clear understanding of the perspectives section is imperative. Discuss the answer to the pre-lab questions with your classmates to ensure that you have a grip on the ideas supporting this lab before proceeding. You will be working with a group and a puzzle kit to demonstrate the structure of DNA and the processes of DNA replication and protein synthesis. Ask questions to clarify these concepts. Sometimes it is relatively easy to take puzzle tiles and organize them into the resulting puzzle without ever appreciating the under lying process or the picture. Materials DNA Replication and Protein Synthesis modeling kit with directions Clear desk space to build models Procedure Activity A. Modeling DNA Structure 1. Follow the instructions provided by your instructor and answer the appropriate questions on your Report Sheet to understand DNA Structure. 2. Once all group members understand the structure of DNA, call your instructor, demonstrate the model, and have them sign Table 2 (page 104) on the Report Sheet. Activity B. Modeling DNA Replication 1. Follow the instructions provided by your instructor and answer the appropriate questions on the Report Sheet to understand DNA Replication. 2. Once all group members understand the replication process of DNA, call your instructor, demonstrate the model, and have them sign Table 2 (page 104) on the Report Sheet. Activity C. Modeling Protein Synthesis = Transcription 1. Follow the instructions provided by your instructor and answer the appropriate questions on your Report Sheet To understand DNA Transcription. 2. Once all group members understand the transcription process of DNA, call your instructor, demonstrate the model, and have them sign Table 2 (page 104) on the Report Sheet. Activity D. Modeling Protein Synthesis = Translation 1. Follow the instructions provided by your instructor and answer the appropriate questions on your Report Sheet To understand RNA Translation. 2. Once all group members understand the translation process of mRNA, call your instructor, demonstrate the model, and have them sign Table 2 (page 104) on the Report Sheet. Activity E: Group Report Sheet 1. As a group complete the questions on pages 103 through 106 and turn in one Report Sheet at the start of the next lab. Biology& 100 Laboratory Manual Mr. Brumbaugh 102 Revised Summer 2016 Report Sheet Lab Section: . Modeling DNA Replication & Protein Synthesis Exercise Group Names: . . . . The following questions should be answered as you build the model to represent DNA, replicate DNA, and synthesize a protein. 1. To which carbon of the nucleotide sugar does the nitrogen base and the phosphate group attach? 2. If the “backbone” of one strand runs 5’ to 3’, what is the orientation of the opposing strand? 3. How are the nucleotides arranged in the DNA molecule? 4. How does DNA replicate and why is maintaining molecular orientation critically important? 5. What is a mutation and how is it reproduced? 6. How does a deoxyribose sugar differ from a ribose sugar? 7. List two ways to tell that this is a model of DNA and not RNA. Biology& 100 Laboratory Manual Mr. Brumbaugh 103 Revised Summer 2016 Noncoding Strand Coding Strand 3’ - C G G T C C A G T C C A - 5’ m-RNA Amino acid sequence of peptide 8. In what molecule(s) do you find the code, the codon, and the anti-codon sequences within their structure? 9. Where is the eukaryotic cellular site of transcription and translation? 10. What could happen to the protein after construction but before it becomes functional? Instructor’s Initials Modeling DNA Structure Modeling DNA Replication Modeling Transcription Modeling Translation Table 2 Instructor’s Initials You need to obtain your instructor’s initials in each box once your group has shown knowledge or each portion of the lab. Application Questions: 1. The DNA sequence in the box on the next page is part of the non-coding strand of the -globin gene (beta globin) that codes for a small portion of hemoglobin, the protein that transports oxygen in your blood. Within the box write the base sequence of the coding strand in the table below - Indicate the 5’ and 3’ ends. Now record the base sequence that would result if this section of the -globin gene were to be transcribed - Indicate the 5’ and 3’ ends. Finally, use the table of the genetic code (Figure 9 and Table 1) to translate this mRNA into protein. List in the table below the order of the amino acids that would be found in the resulting peptide. Biology& 100 Laboratory Manual Mr. Brumbaugh 104 Revised Summer 2016 Non-coding Strand of -globin gene 5’ - A C C C A G A G G T T C T T T - 3’ Coding Strand m-RNA Amino acid sequence 2. The entire -globin chain has 146 amino acids. What would be the minimum number of nucleotides in the mRNA that would encode the -globin protein? Explain your reasoning: 3. How many tRNA molecules will be needed to translate the -globin mRNA into protein? Explain your reasoning: 4. List the base sequence of the anticodon for each of the tRNA molecules needed to translate the -globin mRNA into protein in the table below. Anticodon 1st 2nd 3rd 4th 5th Base sequence in anticodon of tRNA Table 3 tRNA Anticodons Counter match anticodons with the codons in the example mRNA above. 5. Determine the effects of the following mutations on the -globin gene. Use the three letter amino acid abbreviations (Table 1) to write the sequence of amino acids that would result if there was a mutation in which an “A” has substituted for the underlined C of the non-coding strand of the -globin gene. Note: Any changes to the non-coding strand will always affect the coding strand! Base sequence of coding strand of the mutant DNA: . Base sequence of the new mRNA: . Amino acid sequence: . Biology& 100 Laboratory Manual Mr. Brumbaugh 105 Revised Summer 2016 6. What would be the amino acid sequence if an “A” substituted for the underlined G? Base sequence of coding strand of the mutant DNA: . Base sequence of the new mRNA: . Amino acid sequence: . 7. What would the amino acid sequence be if you deleted the underlined C of the non-coding strand? Base sequence of coding strand of the mutant DNA: . Base sequence of the new mRNA: . Amino acid sequence: . 8. What can you conclude about the impact on a protein from these two types of mutations? (I.e. How does a point mutation that involves the substitution of a single base affect a protein compared to a point mutation involving the deletion of a single nucleotide?) Explain the reasoning behind your response. Biology& 100 Laboratory Manual Mr. Brumbaugh 106 Revised Summer 2016 Pre-Lab Report Sheet Lab Section: Modeling DNA Structure, Replication, & Protein Synthesis Exercise . Name: . Before coming to lab carefully read the previous pages on Modeling DNA Structure, Replication, & Protein Synthesis then answer these pre-lab questions. Be prepared to hand in your responses to the pre-lab questions at the start of lab. 1. Explain how it is possible for you to have so many different kinds of cells in your body (e.g. muscle cells, skin cells, liver cells, etc.) when nearly all of the cells contain the same 46 molecules of DNA (chromosomes). 2. Explain in your own words your understanding of the central dogma of biology. 3. Answer each of the following questions. What is the primary structure of a protein? Of what importance is the primary structure of a protein? What ultimately determines the primary structure of a protein? What is a mutation? During what process are mutations most likely to occur? Why do mutations affect the primary structure of a protein? Biology& 100 Laboratory Manual Mr. Brumbaugh 107 Revised Summer 2016 4. Describe in your own words the structure of DNA. 5. Describe in your own words how DNA makes copies of itself. (I.e. Describe DNA replication.). 6. Protein synthesis involves two processes, transcription and translation. Describe in your own words how each process occurs. Transcription Translation Biology& 100 Laboratory Manual Mr. Brumbaugh 108 Revised Summer 2016 Laboratory 9: Critical Thinking and Classification Pre-lab Assignment Before coming to lab carefully read the Perspective and the Introductions on Critical Thinking and Classification (pages 109 &110, 111, 113 & 114). Hurrah, hurrah no pre-lab report. Perspectives Since people first began to question how nature works and how they fit into nature, problems have arisen. Answers to questions relating to man’s relationship with other creatures on the earth posed only more questions. Organizational problems arose when dealing with knowledge. How can we get a common language between investigators to eliminate duplication of effort or simply to communicate our ideas with the vast varieties of native languages? How do we solve problems and organize data became the focal point of science. Methods and processes were needed to allow scientists to communicate worldwide. The process of critical thinking allows scientists and you to evaluate and understand a problem, devise a plan to solve the problem, carry out the plan, and lastly to evaluate the outcomes of the execution of the plan. The following outline expands this process with more detail and could or should be applied to almost any situation with which you could face in your academic life or career. An eighteenth–century Swedish naturalist, Carolus von Linnaeus, is largely responsible for creating the system of scientific names that we use today (Kingdom, Phylum, Class, Order, Family, Genus, and Species (Today we have added Domain before kingdom.)). Linnaeus undertook the formidable task of naming and classifying all plants and animals based primarily on their physical characteristics (Why didn’t he do all the kingdoms?), assigning each organism a two–part name called a binomial. The first word of the binomial designates the group to which the organism belongs; this is the genus name. All oak trees belong to the genus Quercus. Each kind of organism within a genus is given a specific epithet or species. Thus, the scientific name in the Linnaean system for white oak is Quercus alba L. (with the L. identifying the classifier in this case Linnaeus), while that of bur oak is Quercus macrocarpa L. Today we use other criteria such as chemical make-up, DNA similarities, breeding habits, and/or behavioral habits to classify the organisms. The techniques of classification are continually adapted and being re-evaluated as new technology makes scientists aware of different aspects of the relatedness of specimens Exercise A: Critical Thinking and Problem Solving Goals of Lab Exercise To develop some skills in solving problems Learn to make representative drawings of specimens Introduction The following exercise is designed to give the student the opportunity to develop skills in problem solving by following a stepwise procedure. Practicing the procedure is critical in understanding the nuances of problem solving. By critically reading the following outline the skill of solving problems can be developed and put into practice. The application of these skills moves well beyond the scope of this exercise and can give the student a skill that is applicable in other aspects of their life. A General Approach to Problem Solving 1. Understand the Problem. First, you have to understand the problem. What is the unknown? What are the data? What is the condition? Is it possible to satisfy the condition? Is the Biology& 100 Laboratory Manual Mr. Brumbaugh 109 Revised Summer 2016 condition sufficient to determine the unknown? Or is it insufficient? Or redundant? Or contradictory? Draw a figure. Introduce suitable notation. Separate the various parts of the condition. Can you write them down? 2. Devise a Plan. Second, find the connection between the data and the unknown. You may be obliged to consider auxiliary problems if an immediate connection cannot be found. You should obtain eventually a plan of the solution. Have you seen it before? Or have you seen the same problem in slightly different form? Do you know a related problem? Do you know a theorem that could be useful? Look at the unknown! And try to think of a familiar problem having the same or a similar unknown. Here is a problem related to yours and solved before. Could you use it? Could you use its result? Could you use its method? Should you introduce some auxiliary element in order to make its use possible? Could you restate the problem? Could you restate it still differently? Go back to definitions. If you cannot solve the proposed problem try to solve first some related problem. Could you imagine a more accessible related problem? A more general problem? A more special problem? An analogous problem? Could you solve a part of the problem? Keep only a part of the condition, drop the other part; how far is the unknown then determined, how can it vary? Could you derive something useful from the data? Could you think of other data appropriate to determine the unknown? Could you change the unknown or the data, or both if necessary, so that the new unknown and the new data are nearer to each other? Did you use all the data? Did you use the whole condition? Have you taken into account all essential notions involved in the problem? 3. Activate the Plan. Third, carry out your plan. Carrying out your plan of the solution, check each step. Can you see clearly that the step is correct? Can you prove that it is correct? Try to anticipate problems with the plan, but don’t radically change the plan unless an obvious failure. 4. Evaluate the Plan. Fourth, examine the solution obtained. Can you check the result? Can you check the argument? Can you derive the result differently? Can you see it at a glance? Can you use the result, or the method for some other problem? Materials for this exercise Meter Sticks, Cardboard or File Folders and Rulers Pickle Balls Boxes and Scissors String Procedure You will work in groups of four. Be sure to list your lab partners on your write-ups. Your writeups will contain responses to all questions posed in the following exercise to include all calculations and work. I would suggest looking at the perspectives and introduction of this lab for direction in determining how you and your co-investigators proceed. Keep in mind that a clear presentation on your approach to solving the problem is more important than the “correct” answer. Remember there is more than one way to solve any of the exercises and their inclusion in your problem solving approach and presentation will only strengthen your arguments. In some cases you will actually be able to follow through to test your ideas. If you feel you need materials other than what is provided you should ask. Determine the maximum number of pickle balls that would fit in this room. Assume that the room is completely empty (no lab benches, no people, etc.). Do not include the space in the fume hoods or shelves (It’s a box, O.K.). Explain your procedure (What you did?) to solve this problem. Give all measurements and show all calculations. All measurements should be done in the metric system. A bunch of pickle balls, various containers, string, and meter sticks will be provided. Please put your group’s names on separate paper to explain your results to include all of your calculations, your step wise procedure, and turn in next week at the beginning of lab. Biology& 100 Laboratory Manual Mr. Brumbaugh 110 Revised Summer 2016 Exercise B: Classification I Pre-lab Assignment Before coming to lab, carefully read the pages on critical thinking and classification. Goals of Lab Exercise To use problem solving skills to classify inanimate objects Introduction Putting objects into groups because they share some similar characteristic is called classification. Nearly all branches of science use systems of classification. Systems of classification are necessary to help the scientist handle the huge amounts of data and facts that have accumulated over the years. When individual objects are placed into groups based on properties they have in common, the study of a large number of objects is made much easier. In this exercise, you will have the opportunity to attempt many ways to classify a group of objects. Each time you classify an object according to a different aspect of it, you learn more about it. You will learn how classification of a large number of objects into a few categories makes it easier to study the objects, because all the objects in a specific group have something in common. In this exercise you will have a chance to devise your own classification system. You will make your own rules for setting up categories and subcategories. To be a successful classifier, you will have to make use of all your powers of observation, especially your sense of sight and your ability to think. In other experiments, you’ll make use of your other senses as well. Good luck! Be sure to read and use the guidelines above for problem solving. Materials for this exercise Each group will need approximately 100 buttons. Procedure 1. You may work as a group. You will need about 100 buttons selected at random from the button collection. Buttons, of course, are common objects that you see every day. But have you ever stopped to think that buttons could be put into groups because they share certain similar characteristics? Examine the buttons and decide how you want to classify them. Some people form groups based on the sizes, shapes, colors, and other characteristics of the buttons. When you have decided how to group the buttons, you should then form subgroups and further subdivide, so you have at least three levels of classification (see next page for an example). For example, if you choose to group the buttons by size, you can have large buttons, large round buttons, and large white round buttons. 2. After you have classified them, make a chart on a separate sheet of paper showing how your classification scheme works. Use the following example (Figure 1) to guide your creativity, but make it your own. Also list the number of buttons in each group and subgroup. After recording your classification scheme, repeat the experiment using different criteria for the groups. Compare your classification system with those of other people in the class. Return the buttons!! 3. Next take one of your schemes and condense it into a dichotomous key (Figure 2). For instance the accompanying dichotomous key would be appropriate for the sample classification chart (Figure 1). Biology& 100 Laboratory Manual Mr. Brumbaugh 111 Revised Summer 2016 4. Group all of your buttons into the center of the table and invite a fellow scientist from another lab to visit your lab and ask them to use your key to identify any button of their choosing. If they are able to correctly identify the button write their names on your dichotomous key. Do this with 2 other lab groups (three names total). 5. Please describe your strategies of classifying in words and turn in your scheme and key next week with your group names. Sample Classification Chart Classification by Size, Shape, and Color Large Buttons 10 Round Buttons 7 Blue 3 Red 4 Medium Buttons 25 Square Buttons 3 Blue 0 Red 3 Small Buttons 15 Round Buttons 12 Square Buttons 13 Round Buttons 4 Square Buttons 11 Blue 8 Blue 1 Blue 2 Blue 8 Red 4 Red 12 Red 2 Red 3 Figure 1 Classification Scheme Simple classification scheme of buttons. 1a. Buttons all large (>3cm in across) 2a. Buttons round sometimes with many straight edges approx. a circle 3a. Buttons blue 3b. Buttons red 2b. Buttons all square or rectangular 4a. Buttons blue 4b. Buttons red 1b. Buttons other than large 5a. Buttons medium (1.5–3 cm in across) 6a. Buttons round sometimes with many straight edges approx. a circle 7a. Buttons blue 7b. Buttons red 6b. Buttons all square or rectangular 8a. Buttons blue 8b. Buttons red 5b. Buttons small (<1.5 cm in across) 9a. Buttons round sometimes with many straight edges approx. a circle 10a. Buttons blue 10b. Buttons red 9b. Buttons all square or rectangular 11a. Buttons blue 11b. Buttons red Figure 2 Dichotomous Key Simple dichotomous key based on a scheme of buttons. Biology& 100 Laboratory Manual Mr. Brumbaugh 112 Revised Summer 2016 Exercise C: Classification II Pre-lab Assignment Before coming to lab, carefully read the pages on critical thinking and classification. Goals of Lab Exercise To use classification skills to classify specimens Develop skills using dichotomous keys to identify specimens Learn to make representative drawings of specimens Introduction At the beginning of any serious study, it is important not only to learn precisely which species are present, but to be able to recognize them at different stages of development. The time is not long past when the field biologist felt well satisfied with their work after presenting a mere list of the dominants. It is now widely acknowledged as poor policy to learn only a few of the dominants and neglect the remaining flora and fauna. (Remember the snail darter or how about the spotted owl?) Subordinate and even rare species often have much value for indicating special conditions (present and past) and they sometimes foretell the future. All species have some indicator significance whether it is known at present or not. Maybe a certain species indicates that Douglas fir will eventually grow at a particular site or that 350 years from now the Douglas fir will be replaced by spruce and hemlocks. We are all great classifiers, already. Every day, we consciously or unconsciously classify and categorize the objects around us. We recognize an organism as a cat or a dog, or an aardvark, or an oak tree. But there are numerous kinds of oaks, so we refine our classification, giving the trees distinguishing names such as “red oak,” “white oak,” or “bur oak”. These are examples of common names, names with which you are probably most familiar. Scientists are continually exchanging information about living organisms. But not all scientists speak the same language. The common name “white oak,” familiar to an American, would probably be unfamiliar to a Spanish biologist, even though the tree we know as white oak may exist in Spain as well as in our own backyard. Moreover, even within our own language, the same organism may have several common names. For example, within North America, a “gopher” may also be called a “ground squirrel,” a “pocket mole,” or a “groundhog”. On the other hand, the same common name may actually describe many different organisms; there are more than 300 different trees called “mahogany”! To circumvent the problems associated with common names, biologist use scientific names that are unique to each kind of organism and that are used throughout the world. Taxonomy is the science of classification (categorizing) and nomenclature (naming). For the purposes of this class we will separate all living organisms into five kingdoms, recognizing that other classification schemes exist and are being developed and discussed in scientific circles today. The kingdoms are outlined below. What criteria would you have used to delineate these groupings? Kingdom Monerae (prokaryotic organism) Kingdom Protistan (euglenoids, chrysophytes, diatoms, dinoflagellates, slime molds, and protozoans) Kingdom Myceteae (fungi) Kingdom Plantae (plants) Kingdom Animalae (animals) Biology& 100 Laboratory Manual Mr. Brumbaugh 113 Revised Summer 2016 Let’s consider some examples of the scientific system of classification using some common species: Category Kingdom Phylum (animals) or Division (Plants) Subphylum Class Order Family Genus Specific epithet Domestic Cat Animalae Chordata Common Buttercup Plantae Anthophyta Spider lily Plantae Anthophyta Vertebrata Mammalia Carnivora Felidae Felis silvestris Magnoliopsida Ranunculales Ranunculaceae Ranunculus acris Liliopsida Liliales Liliaceae Hymenocaulis carbaea Since the naming of things is so important for study and communicating what you are studying you need to be able to identify each organism you meet. How do you do that? You start young. You start excited. You start now, using a dichotomous key that allows you to identify organisms. Keys are based on dichotomous questions, meaning they give you either one choice or another, but sometimes they are not. You will get to work with both kinds before your biological safari ends. Materials for this exercise Dichotomous keys and Specimens Procedure Your team will divide and complete the following assignments and turn in one packet at the beginning of the next lab along with the previous assignments. 1. Using the descriptions of various phyla in the Kingdoms Plantae and Animalae Highlights in Appendix G, each group will go out in the wilds and find a creature and identify it to the level of Division or Phylum. Draw the critter to scale (Appendix D). Turn both your drawing and identification in with the report sheet at the beginning of your next lab period. 2. Option 1: Complete the "Classification Table” (Table 1). Observe the specimens at each station and identify the kingdom, phylum, and common name for each organism to complete the columns using the provided keys, only fill-in symmetry for Animalia specimens. 3. Option 2: Using your text or other sources answer the questions on the report sheet pages 116 and 117. 4. Complete all the pages of the report sheet and turn in next week along with strategy and calcualtions for filling the room with pickle balls, your classification schemes and dichotomous key for the buttons, and your critter drwawing as a group. Biology& 100 Laboratory Manual Mr. Brumbaugh 114 Revised Summer 2016 Report Sheet Lab Section: . Critical Thinking & Classification Exercise Group Names: . . . . Option 1: Station Kingdom Phylum Common Name Symmetry 1 2 3 4 5 6 7 8 9 10 11 12 Table 1 Classification Table This table is used for student classifications based on their observations of various organisms presented in lab. Biology& 100 Laboratory Manual Mr. Brumbaugh 115 Revised Summer 2016 Option 2: Classification Questions Taxonomy 1. Define radial and bilateral in terms of animal symmetry. 2. Why do we have a classification system? Is this system fixed? 3. Name the seven general categories used in this system. 4. What is the conventional way of writing a specific genus and species? Systematics 1. In a few words identify what topics the study of systematics addresses. 2. Complete this table: Symmetry type Invertebrate representative or example radial bilateral no symmetry 3. Respond to this statement: The body cavity is also called the gut. 4. During embryonic development in humans, the first opening becomes the 5. During embryonic development in mollusks, the first opening becomes the Biology& 100 Laboratory Manual Mr. Brumbaugh 116 Revised Summer 2016 Phylogeny 1. In your own words define phylogeny. 2. Complete this table: Phylum Two examples Two characteristics Arthropoda Molluska Echinodermata Chordata Your choice: Name two other animal or plant Phyla or Domain plus cite 2 characteristics of each category that you name. 1. Biology& 100 Laboratory Manual Mr. Brumbaugh 2. 117 Revised Summer 2016 The Final Biological Doodle Page Biology& 100 Laboratory Manual Mr. Brumbaugh 118 Revised Summer 2016 Laboratory 10: Paper Project Perspectives Each person will work in a group of about 4 people (2-4) on a project that addresses a topic related to biology. It can be a scientific question or a question arising from the application of scientific advances to societal problems. For this assignment, the topic must have some interesting biology associated with it. When presenting various aspects of the issue it is important that there is a biological basis to support the various aspects of the topic. Ethics are important, but arguments that only deal with what is moral or ethical are not sufficient. The group will select a topic (see page 123 of this manual for ideas) and then divide the topic into four subcategories or topics. For example the group decides to investigate Stem cell research. They could then divide up the overarching topic into the history of stem cell research, definition and discussion of the process of stem cell research, the protagonist’s side of stem cell research, and the antagonist’s side of stem cell research. Each student will find their own references, write a four page narrative summary on their individual topic, and finally the group will organize an oral presentation to present their research to their lab section. The Paper Project consists of four graded assignments: Project References, Initial Draft of Position Paper, Final Draft of Position Paper, and a Group Oral Presentation. Each of these four assignments is described on the pages that follow. Refer to the table below for dues dates and how each assignment contributes towards your quarter grade. Assignment Basis for Grade % of Grade for Paper Due Dates for 1. Project One list per person 10% (10 points) At the start of your lab during References (Individual grade) week 5 2. Initial Draft of One paper per person 30% (30 points) At the start of your lab during Position Paper (Individual grade) week 7 3. Final Draft of Position One paper per person 60% (60 points) At start of your lab during Paper (Individual grade) week 9 4. Group Oral One plan per group 70 points At start of your lab during Presentation (Group grade) either week 9 or 10 Table 1 Paper Assignment Table This table shows the due dates for each aspect of the paper project. This assignment is to give you the opportunity to use the knowledge you have gained or will gain throughout this course and kind of bring the ideas to bear by presenting a body of evidence to support your premise. Approach this project with the vigor of attempting to move your peers into a realm of questing knowledge. Procedure Assignment 1: Project References = 10 points Each group member will individually: 1. Turn in a typed list of the references, with at least 4 being referred, they have found. The list should be in the proper format outlined in Appendix C. 2. Each reference page should have a typed working title and the authors associated with the paper. Biology& 100 Laboratory Manual Mr. Brumbaugh 119 Revised Summer 2016 Using the Holman Library (See Appendix F) The GRCC Library offers excellent electronic methods for finding reference materials for your projects. Most can be accessed directly via the World Wide Web. The GRCC Library Catalog contains all of the books at the college, but books are probably not the best source for this project. Most of your references should be periodicals as they are usually more specific and up-to-date. There are several searchable electronic databases available for your use. A GRCC librarian will be happy to assist you in determining the databases most appropriate for your project. You may find that some of the periodicals that would be of greatest help for your project are not available through the GRCC Library. If this is the case, please talk to a librarian about how to get copies of articles that you need from other libraries in the region. Often you can get what you need in a couple of days. The references you pick are not all created equally! The best source is information from the scientists who conducted the study. Try to find information of this type—much of it may be too technical but you should be able to glean some information from it. The second best source is popular science journals such as Scientific American, Science News, American Scientist, or Discover. Most of your references will probably be of this type. Since these periodicals are devoted to science, they tend to be better sources of information than general magazines such as Time or Newsweek. General popular references such as newspapers and general magazines may sometimes be helpful but don’t limit yourself to these. Each group should try to find at least 4 references of high quality. Remember that part of your grade for your paper is based on the quality of your references. The World Wide Web also provides an excellent source of materials. Web pages vary in quality enormously, so you should take care to use sources that provide accurate information. Look carefully for the biases of the authors. Many news magazines, newspapers and journals now publish on the web. These will tend to be more reliable than individually published web pages. The latter may be very useful, though, particularly if they cite references. Do not limit yourself to material that is strictly web based. Every scientific publication provides an “Instructions to Authors” that describes the format for the references section and all other requirements for papers they will accept. The format for citing references varies slightly from one scientific publication to another. By following the guidelines as outlined in Appendix C (How to cite references.) you will insure your citations are cited correctly. Assignments 2 & 3: Draft and Final Position Paper = 30 and 60 points respectfully Every member will turn in a paper on one aspect of your group’s issue or investigation using the references you have found. This report should be about 4 pages typed and double-spaced. Your title page (containing the title, author, course # and section, due date, and Instructors name), figures, and references should be on additional pages. High quality papers are expected. Use a word processor and save your electronic version of the paper until after you receive your grade. Computer crashes are not an excuse for late papers. It’s a good idea to keep a hard-copy too. The initial draft of your paper is due at the beginning of lab during the 7th week. You should bring 2 copies of your paper to lab. One copy will be turned in and the second copy will be given to another member of the class. You will read another class member’s paper, make comments on the paper, and return it to the writer after signing the title page. The final version of your paper is due at the start of lab during the 9th week of lab. You will receive two grades for your paper: a rough grade and a final grade. The rough paper grade will be based on the quality of your rough draft of your paper. The final draft of your paper will be graded more carefully. You should turn in your rough paper (with comments) and my grade rubric with your final paper. One of the goals of this course is for you to be able to analyze and form an informed opinion about issues related to cellular biology and genetics. Issues are questions about which informed people disagree. Issues involve ideas that can be controversial and there is no right or wrong solution, but your opinion needs to be supported by evidence and must also have some interesting biology associated with it. When presenting various sides of an issue it is important that there is a biological Biology& 100 Laboratory Manual Mr. Brumbaugh 120 Revised Summer 2016 basis to support the various sides. Ethics are important, but arguments that only deal with what is moral or ethical are not sufficient. Your paper should have three main components: problem posing, problem solving and persuasion. It is important that all three parts are not just present but are presented clearly and persuasively. And remember that the clarity of your writing is important, including organization, spelling and grammar. Use the following information to assist in the organization of your paper. 1. Problem Posing The topic should be clearly and explicitly stated in the introduction and remain as a focus through the entire paper. Part of your job is to convince the reader that this is an important issue and everyone should be concerned about it. 2. Problem Solving and Persuasion Convince the reader that your opinion is correct, even if it falls somewhere between the extreme positions. You should support your opinion with evidence from your readings. Indicate the source of your evidence by using “in-text citations” (Appendix C), and then put a list of references at the end of the paper. Indicate the arguments from other sides of the issue. Give references in the format outlined in Appendix C. Tell us why these arguments shouldn’t convince us. In other words, you need to present all sides of the issue but convince us to agree with you. Most issues are not black or white so you may fall somewhere between the extreme positions. If that is the case then indicate what evidence is most convincing and what evidence is still weakly supported. Indicate what further evidence you would like to see to solidify your position. What does this problem and solution have to do with me? And/or what is its general significance to the world? This should sum up your essay and leave the reader thinking: “Wow, that is an important conclusion and I should act on it in some way.” You should include a reference section at the end of the paper. We will evaluate the quality and quantity of your references. Place the references in alphabetical order by author and use proper format. Assignment 4: Group Oral Presentation = 50 points The final component of this assignment is the group oral presentation (see Appendix F). This should be a means for your group to communicate your new found knowledge to your peers in (hopefully) a convincing manner. Each member will be involved in the presentation by presenting a united front of ideas to the audience. What material to present after all, a presentation is meant for conveying information? You need to know the topic as a whole, as well as the specific aspects of it. For example, if you were giving a presentation on vaccines against HIV, you need to have a thorough knowledge of the HIV life cycle and the human immune system’s response to HIV, as well as a specific knowledge about how vaccines against HIV might work and why they so are controversial. How to organize the material; organized information is easier to remember for you and easier to understand for others (Use the rubric in the syllabus for assistance). Notes are fine, but don’t write a paper—an organized outline or a list is much more useful for a presentation. IMPORTANT: Eye contact with the audience is essential—do not read directly from your notes, PowerPoint presentation, etc.—use them only as quick reminders as to what you want to discuss—do not use them as a “crutch,” only as an occasional aid. How to present the information; there are many ways to present material. The best format is the one that allows you to convey information clearly. A controversial topic might involve a debate format, and statistics might be presented best graphically, etc. Use visual aids to facilitate the audience’s understanding of your presentation. You can use PowerPoint, overhead projector transparencies, video clips, etc. I can help you use these, but only if you notify me well in advance of your presentation. Biology& 100 Laboratory Manual Mr. Brumbaugh 121 Revised Summer 2016 Your group’s presentation should be about 20 minutes long. This works out to about 4 min. per person per group and leaving additional minutes for questions/class discussions. You may incorporate various styles; debate, skit, lecture, poster presentation, etc., or you can stick to one style. Do not to read a prepared paper or lengthy note cards. DO NOT give too much information, but, rather, summarize the important points in a thoughtful manner. Go slowly, and emphasize main points. Use visual aids to facilitate the audience’s understanding of your presentation. You can use PowerPoint, overhead projector transparencies, video clips, etc. I can help you use these, but only if you notify me well in advance of your presentation. How to get started! Search existing literature, start early because searches take time (See Appendix F). You need to know what information is available, as well as hot or controversial topics in the fields. To gain a comprehensive view of the field, I recommend starting with a book chapter or a review article. Use the reference sections from those to find more detailed information. Moreover, there are many links at the class website that may prove useful. Come talk to me in person. You can get a lot of feedback from me at any point during the preparation. Added benefit is that you can figure out my preliminary evaluation of your presentation, so that you will know how much and what kind of work you have to do quality work. Organize your work. You are working with others. Clearly organizing and designating responsibility for each is extremely important. I recommend getting together regularly (e.g. 2-3 times week for at least 30 min. each), so that you can give each other updates on how things are going. Your group will be scored by the rubric found in your syllabus for the group presentation. Biology& 100 Laboratory Manual Mr. Brumbaugh 122 Revised Summer 2016 Possible topics for the paper: Biological Basis for Human Races: Is there a biological basis for dividing people into races? Genetic Modification of Food Crops: Are GM foods safe to eat? Genetic Engineering of Organisms (e.g. plants, animals, or microbes): Do the benefits outweigh the possible drawbacks? The Puzzle of Hypertension in African-Americans: Why is high blood pressure the leading cause of health problems among black Americans while the people of western Africa have among the lowest rates of hypertension anywhere in the world? Somatic Cell Gene Therapy: Should somatic cell gene therapy be used to treat genetic diseases? Slowing Human Aging: Is it possible to slow down the aging process? Genetic Basis of Aging: How important are genes in determining life expectancy? Alzheimer's disease: Is a cure imminent? Why are more women than men affected by it? Abortion Pill: Is the use of RU 486 harmful to woman's health? Should it be banned? Attention-Deficit Hyperactivity Disorder: Is there are genetic basis to the neurological abnormalities involved with ADHD? Hormone Replacement Therapy: Should postmenopausal women use hormone replacement therapy (HRT) to reduce/prevent osteoporosis? Human Cloning: Should human cloning research be allowed/funded by the federal government? Human Fetal Tissue Research: Should the federal government allow/fund medical research involving human fetal tissue obtained from aborted fetuses and umbilical cords or are there alternative sources for stem cells for medical research? Homosexuality: Is there a genetic basis for homosexuality? Thrill/Novelty Seeking: Is there a genetic basis for thrill or novelty seeking? Obesity: Is there a genetic basis for obesity? Genetic Basis of Heart Disease: Are national differences in rates of heart disease environmentally or genetically caused? What is the role of a dietary cholesterol and fat in heart disease? Alcoholism/Substance abuse: Is there a genetic basis for alcoholism/substance abuse? Alternative Cancer Therapies: Traditional (chemotherapy and radiation) vs. alternative/experimental therapy do cancer patients have an alternative to the devastating effects of chemotherapy and radiation therapy? Safety of Food Additives: Do food preservatives/additives pose a significant health risk (e.g. cancer, developmental problems, etc.)? Are they being regulated properly? Hormone use by the food industry: Is it a human health hazard to eat food products derived from hormonally treated animals? Depression: What is the biological cause of depression? Child Abuse: Should mothers of drug-addicted babies/fetal alcohol syndrome be prosecuted for child abuse? Cloning for Medicine: Hype or a possible reality? Genetic Basis of Athletic Performance: Can anyone become a world class athlete if they train properly? What role(s) do genes of the athlete play? Organic vs. “traditional” foods: Do the potential benefits of organic foods outweigh the extra costs involved? Nutritional supplements: Is it worth the expense to take nutritional supplements? (e.g. Vitamin supplements, melatonin, anti-oxidants, etc.) Genetic Testing and Screening: Should widespread testing for cystic fibrosis (or other genetic diseases) be implemented? Biology& 100 Laboratory Manual Mr. Brumbaugh 123 Revised Summer 2016 Appendix A: How to graph scientific data? Often the first step in analyzing the results of an experiment is the presentation of the data in the form of a graph. A graph is a visual representation of the data, which assists in bringing out and finding the possible relationship(s) between the independent and dependent variables. Examination of a graph makes it much easier to see the effect the independent variable has on the dependent variable(s). Accurate and clearly constructed graphs will assist in the interpretation and communication of your data, and when presenting a well-documented argument supporting or falsifying your hypothesis in the final steps of a scientific investigation. All graphs should be easy to interpret and labeled fully. The following guidelines will help you construct a proper graph. Graphing tips 1) Use graph paper of a high quality. 2) A ruler should be used to draw axes and to plot data neatly and accurately. 3) Always graph the independent variable on the x-axis (horizontal axis), and the dependent variable on the y-axis (vertical axis). 4) The scales of the axes should be adjusted so that the graph fills the page as much as possible. The axes often, but not always, start at zero. Choose your intervals and scales to maximize the use of the graph paper. Intervals should be logically spaced and easy to interpret when analyzing the graph (e.g. intervals of 1’s, 5’s, or 10’s are easily interpreted, but non-integer intervals (e.g. 3.25’s, 2.33’s, etc.) are not. To avoid producing a graph with a lot of wasted space a discontinuous scale is recommended for one or both scales if the first data point is a large number. Simply add two tic marks between the zero and your lowest number on one or both axes to show that the scale has changed. 5) Label both axes to indicate the variable and the units of measure. Write the specific name of the variable. Do not label the axes as the dependent variable and independent variable. Include a legend if different colors are used to indicate different aspects of the experiment. 6) Graphs (along with drawings and diagrams) are called figures and are numbered consecutively throughout a lab report or scientific paper. Each figure is given a number, a title that describes contents, and an informative sentence giving enough information for the figure to be understandable apart from the text (e.g. Figure 1 Temperature and Leaf Color Change The relationship between the change in vine maple leaf color and changes in ambient temperature). Generally, this information is placed below the figure or graph. 7) Choose the type of graph that best presents your data. Line and bar graphs are the most common. The choice of graph type depends on the nature of the variable being graphed. Line Graphs are used to graph data that only involves continuous variables. A continuous variable is capable of having values over a continuous range (i.e. anywhere between those that were measured in the experiment). For example, pulse rate, temperature, time, concentration, pH, etc. are all examples of continuous variables (Figure 1). Making Line Graphs 1) Plot data as separate points. Make each point as fine as possible and then surround each data point with a small circle. If more than one set of data is plotted on the same graph, distinguish each set by using circles, boxes, triangles, etc. 2) Generally, do not connect the data points dot to dot. Draw smooth curves, or if there appears to be a linear relationship between the two variables, draw a line of best fit. Biology& 100 Laboratory Manual Mr. Brumbaugh 124 Revised Summer 2016 3) If more than one set of data is plotted on a graph, provide a key of legend to indicate identify each set. Label the graph as a figure; give it an informative title, and a descriptive sentence. Figure 1 pH Effects on Lactase Note that a line graph was used to graph the data because both variables, pH and the rate of digestion, are continuous variables. Bar Graphs are used if the data involves a discrete variable (non-continuous variable). A discrete variable, unlike a continuous variable, cannot have intermediate values between those measured. For example, a bar graph (Figure 2) would be used to plot the data in an experiment involving the determination of chlorophyll concentration (chlorophyll concentration is a continuous variable) found in the leaves of different tree species (The discrete variable is the species of tree). Bar graphs are constructed using the same principles as for line graphs, except that the vertical bars are drawn in a series along the horizontal axis (i.e. x-axis). In the example below, a bar graph was used to graph the data because tree species is a discrete variable since it is impossible to have a value or species between those used. Figure 2 Chlorophyll Concentrations The chlorophyll concentrations were measured mg/grams of leaf in the leaves of three tree species. Biology& 100 Laboratory Manual Mr. Brumbaugh 125 Revised Summer 2016 Appendix B: How to convert to the metric system? Larger Unit Tips for Metric Conversion: 1. When converting from a larger 1 km = 103 m = 1000 m unit of measure to a smaller unit of measure (e.g. from kilometers, 1 m = 100 m km to meters, m) move the decimal to the right. This results -2 1 cm = 10 m = 0.01 m in a larger number. 2. When converting from a smaller 1 mm = 10-3 m = 0.001 m unit of measure to a larger unit of measure (e.g. from m to km) 1 m = 10-6 m = 0.000001 m move the decimal to the left. This results in a smaller number. 1 nm = 10-9 m = 0.000000001 m 3. See below to determine how many decimal places to move. Smaller Unit Figure 4 Metric System Relationships This figure shows the conversion relationships of common metric measurements. Determination of the number of decimal places to Move The number of decimal places moved is equal to the magnitude difference between the exponents of the two units of measure. The exponent scale below illustrates the relationship between exponents. -6 m -5 -4 -3 mm -2 -1 cm 0 m 1 2 3 km 4 Examples 1. 9.25 km =?? mm km to mm is a large to small unit conversion, so the decimal must move to the right. The magnitude of difference between the exponents of each unit of measure is 6: km = 103, mm = 10-3; Therefore: 3 - (-3) = 6 So the decimal place moves to the right six places giving 9,250,000 mm (or 9.25 x 106 mm) 2. 450 µm =?? mm µm to mm is a small to large unit conversion, so the decimal must move to the left. The magnitude of difference between the exponents of each unit of measure is 3: µm = 10-6, mm = 10-3; Therefore: -3 - (-6) = 3 So the decimal place moves to the left three places giving 0.45 mm Biology& 100 Laboratory Manual Mr. Brumbaugh 126 Revised Summer 2016 Appendix C: How to write scientific papers? Scientific papers follow a specific format in order to separate the different aspects of any study or experiment. This allows for lucid presentation of ideas, and facilitates critical evaluation of papers. The format below is not set in stone; it is meant to provide a structural guideline for writing your papers. See journals in any field of environmental science for examples of variations on this nearuniversal scientific writing template (note that Science and Nature are exceptions to this rule). You will find that writing this way may take some getting used to, but helps you to present your work more clearly and perhaps even to think more clearly about the work you have done. All papers should be typed, double-spaced (except the abstract), with at least one-inch margins on all sides. Any statements not original to you should be properly cited in the text using the scientific citation style, and listed in the section called Literature Cited at the end of your paper in the style below. Title Page The title page is the first page of the paper and should contain the following: An informative title The full names of all group members Course number Instructor’s name Your lab day and time Due date for the paper A good title is informative, i.e. it summarizes as specifically, accurately, and concisely as possible what the paper is about. For example, if you were investigating the effect of temperature on the feeding preferences of a certain type of caterpillar found on tobacco plants, acceptable titles might be “Effect of Temperature on the Feeding Preferences of the Tobacco Hornworm Larvae, Manduca sexta”, or “Does Temperature Influence which Diet the Tobacco Hornworm Larvae, Manduca sexta, will Select? The following titles would be uninformative and too general: “Effect of Temperature on Caterpillars”; “How Temperature Affects the Tobacco Hornworm Larvae, Manduca sexta”; “What is the Preferred Diet of the Tobacco Hornworm, Manduca sexta?” Abstract (optional for this class) Present a concise statement of the questions, general procedure, basic findings, and main conclusions. This is a brief, all-encompassing section summarizing what you discuss in the rest of the paper, and should be written last, after you know what you have said! This section only should be written as one single-spaced paragraph not to exceed 200 words. Introduction Present a background for the work you are doing and put it into an appropriate context (e.g. scientific principles, environmental issues, etc.). Cite any references in the text you used as sources for your background Information. What questions are you asking in your study? What organisms or ideas were studied and why are they interesting or relevant for your study? Identify the subject(s) and clearly state the hypotheses of your work. Tell the reader why he/she should keep reading and why what you are about to present is interesting. Briefly state your general approach or methods (e.g. experimental, observational, computer simulation, a combination of these, etc.) as a lead-in to the next section. As a general rule the introduction section should be about the length of the discussion section. Most introductions are designed to compel the reader to read the article not bog down the reader with to much detail. Biology& 100 Laboratory Manual Mr. Brumbaugh 127 Revised Summer 2016 Materials and Methods Describe the equipment used in your study. Explain the methods in paragraph format used to answer your questions in sufficient detail that someone else could repeat the work. Cite alreadypublished methods (e.g. lab or computer manual or handouts) but describe any modifications, avoiding lengthy explanations. Briefly explain the relevance of the methods to the questions you introduced above (e.g. "to determine if light limited algal growth, I measured...."). This section may be subdivided (with subheadings) to describe distinct parts of your study or experiment. If appropriate, include a description of the statistical methods you used in your analysis. Results Present in an orderly fashion what you discovered in this study (this may be subdivided as above). Describe the results in text and if appropriate, present them also as tables or graphs, by referring to the table of graph by number in the text. Graphs and tables should be numbered and titled for clear reference in your results and discussion sections. Be sure to label both axes of all graphs (e.g. growth rate, height, number of species, water consumed, etc.) and include units (e.g. meters, gallons, seconds, etc.). Graphs should be understandable on their own without reading the text, and be accompanied by a brief, informative caption explaining what each graph or table shows. In referring to your results, avoid phrases like 'Table 1 shows the rate at which students fall asleep in class as a function of the time of day that class is taught”. Rather, write: "Students fall asleep in class twice as frequently during evening than day classes (Table 1)”. The results section should avoid discussion and speculation. This is the place to tell the reader what you found out, not what it means. Discussion Explain your results in detail, speculating on trends, possible causes, and conclusions. What conclusion can be drawn from your results? Present major findings first, then minor ones, including any natural history descriptions or other background information that may not be necessary, but would help fill in the bigger picture. Compare your results with those of other workers and cite the references you used for comparisons. Put your results in the context of the hypotheses and other material in your Introduction. Where do your data fit in to the big picture? What problems arose in your study and how could they be avoided in the future? If your results were not consistent with the predictions you made (what you thought would happen before you did your study, based on a specific hypothesis or other background information) why do you think this is the case? Explain any exceptional aspects of your data or unexpected results. Examine your results for possible error or bias. Refer to your results to solidify your ideas. Where does your study lead? Here you may recommend further work that could augment the results of the study you have presented. What are your major conclusions? These ideas are what make a discussion interesting and thought provoking for your reader. Acknowledgements (optional) In this section you should thank anyone who has helped you in any aspect of this project, funding agency, or whomever. (e.g. "I thank Claudia Mills for help with the computer program, Milo Lee for reading my electric meter, Al Gore for counting cockroaches, and Mike Kalton for valuable discussions of the Ideas underlying these data.). Literature Cited In this section you list only the sources that you have actually cited in the paper, either as general background or specific examples. It is not an exhaustive bibliography. Use the citation style below. Biology& 100 Laboratory Manual Mr. Brumbaugh 128 Revised Summer 2016 How to Cite Sources in Scientific Writing In-Text Citations There are typically not footnotes or endnotes in scientific writing as there are in humanities and the social sciences. Instead, all citations occur in the text in parenthetical format, with the author(s) and date of publication. Use the following as an example: Parsons (1996) found that naked mole rats dig six times faster in desert soils than dung beetles dig through dung. Alternatively, Naked mole rats dig six times faster in desert soils than dung beetles dig through dung. (Parsons 1996). Or, Naked mole rats dig six times faster in desert soils than dung beetles dig through dung. (1) This notation (1) refers the reader to the bibliography page which is sequentially numbered and each citation from this author is referred to in this fashion. It's that simple! Be sure to list any sources you cite in the text in the Literature Cited section, and only those that you cite. As a rule of thumb, if there is more than one author of a source, simply use the first author's last name, followed by et al. (e.g. [Parsons et al. 1996]). This is Latin for "and others". The complete list of authors will appear in the full citation at the end of your paper. Literature Cited or Bibliography Your Literature Cited should appear in alphabetical order by first author, and by year if there are multiple sources by the same author(s). Underline journal and book titles, but not the titles of individual articles in journals or edited (multi-authored) books. Use the following as examples for citing various kinds of sources (with thanks to M. Weis): Citing Journal and Magazine Articles Format Author(s). Publication year. Article title. Journal title volume: pages. Examples Smith, D.C. and J. Van Buskirk. 1995. Phenotypic design, plasticity and ecological performance in two tadpole species. American Naturalist 145: 211-233. Ahlberg, P.E. 1990. Glimpsing the hidden majority. Nature 344: 23. Epel, D. and R. Steinhardt. 1974. Activation of sea-urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. (USA) 71: 1915-1919. Citing Sites on the Internet Often electronic sources are a challenge to cite because they often lack critical information. You should do your best to provide as much of the following as possible. The complete web address should be presented so that anyone else could easily visit the same website. Attempt to include the following elements (not all elements appear on all Web pages): 1. author(s) (last name, first initial) 2. date created or updated 3. title of the page 4. title of the complete web site (if different from the page) 5. URL (full web address) 6. the date accessed. Biology& 100 Laboratory Manual Mr. Brumbaugh 129 Revised Summer 2016 Format Author's last name, First initial. (date created or updated). Title of the page. Title of the complete site. [Online]. Available: http://full.web.address. [Date accessed]. Example Hammett, P. (1997). Evaluating web resources. Ruben Salazar Library, Sonoma State University. [Online]. Available: http://libweb.sonoma.edu/Resources/eval.html. [March 29, 1997]. Citing Books Format Author(s). Publication year. Book Title, edition if known. Publisher, Place of publication, number of pages. Example Purves, W.K., G.H. Orians and H.C. Heller. 1995. Life: The Science of Biology, 4th edition. Sinauer Associates, Inc., Sunderland, MA, 1195 pp. Citing Book Chapters Format Author(s). Publication year. Chapter title. In: Book title (Author(s)/editors, first name first) Place of publication, pages. Example Jones, C.G. and J.S. Coleman. 1991. Plant stress and insect herbivory: Toward an integrated perspective. In: Responses of Plants to Multiple Stresses (H.A. Mooney,W.E. Winner & E.J. Pell, editors), Academic Press, San Diego, pp. 249-280. Citing Newspaper Articles Format Author(s). Date (Year/Month/Day). Article title. Newspaper title Section: Page: Column. Example Bishop, J. E. 1982 November 4. Do flies spread ills or is that claim merely a bugaboo? The Wall Street Journal 1: 1: 4. Williams, M. 1997 January 5. Teaching the net. Seattle Times C: 1: 2. Citing Newspaper Articles with no Identifiable Author Format Anonymous. Date (Year/Month/Day). Article title. Newspaper title Section: page: column. Example Anonymous. 1977 September 6. Puffin, a rare seabird, returns to where many were killed. The New York Times 3:28:1. Citing a Video Format Title of video (videocassette). editor or director. Producer’s name, producer. [Location of Production]: Organization responsible for production, Year. Example New horizons in esthetic dentistry (videocassette). Wood, R. M., editor. Visualeyes Productions, producer. [Chicago] : Chicago Dental Society, 1989. Biology& 100 Laboratory Manual Mr. Brumbaugh 130 Revised Summer 2016 Citing a Government report Format Author/Agency (if no author). Publication year. Title. Publisher, Place of publication, number of pages. Example Mitchell, R.G., N.E. Johnson and K.H. Wright. 1974. Susceptibility of 10 spruce species and hybrids to the white pine weevil (= Sitka spruce weevil) in the Pacific Northwest. PNW-225. U.S. Department of Agriculture Forest Service, Washington, D.C., 8 pp. Biology& 100 Laboratory Manual Mr. Brumbaugh 131 Revised Summer 2016 Appendix D: How to Draw or Make a Scientific Plate or Drawing? Why draw? It is not the objective of this course to “make you into an artist” or even to have you produce drawings of professional quality, even though many of you are capable of such work. It is expected, however, that each student will produce clear and accurate drawings of the organisms and structures that she/he actually observes. Drawing is a tool that will help you focus on what you are really seeing at deeper level that maybe you have seen before. Drawing is a tool that will draw you closer to the body plans of organisms and maybe for the first time open up to you a whole new world. Seeing things for the first time the way they really are is very exciting. It’s amazing how many times we look at something but don’t really see it. Drawing “makes it so.” Drawing will make the vocabulary of science more tangible and available to you. What is bilateral symmetry in the sagittal plane of the anterior section of pineal glands of a frog really mean? Does it mean something special for frog growth and development or is it just an accident. The metaphase plate formed during mitosis in root tips can actually be visualized bringing the boring humdrum words from the text into a vibrant exciting reality, right now! Drawing will show you how things work. The functionality of different parts will be revealed. As you look at a filter feeding daphnia filtering it’s dinner from its aqueous environment you will discover why it is shaped the way it is shaped and “oh yeah” what those parts are for. Those parts will become more than just parts to you. You will find, as the quarter progresses that it becomes easier to make good, acceptable drawings as you develop your powers of observation and become familiar with certain basic drawing techniques. You will also discover that the preparation of these drawings is an excellent way to study specimens, for in order to make acceptable drawings you must observe in detail the form, structure, and interrelation of parts of the object being drawn. Your drawing will record observations clearly and concisely that would require several pages of descriptions to duplicate. Further, you will find your drawings to be excellent review materials, especially since many of the living organisms studied in the laboratory will available to you only during certain laboratory periods, and your drawings will be the only record of personal observations that you have. You will be permitted to use these drawings for study purpose. Make all your drawings directly from the specimens or slides and complete each drawing in the laboratory. Do not merely copy drawings of your neighbor or plates from the textbook. Textbook illustrations are often idealized, or may represent different species from those you are studying in the laboratory and thus do not look like your specimen. Procedure 1. Before you do anything else, study the material to be drawn. From what angle are you going to prepare the drawing? Frequently, the laboratory instructions often direct you to prepare a drawing from a specific viewpoint, such as a cross section of the sagittal plane of the anterior section of a pineal gland. Often times the angle that is chosen depends on specific functions to be studied or maybe just a key feature used in identification of the species. 2. Notice the outline of the entire object to be drawn? 3. Notice what structures are present (how many, how many different kinds). 4. Notice how the structures are interconnected or interrelated? 5. Notice everything that might help you draw the critter or critter part that is in front of you, like; folds and creases connection shapes holes, hairs, and hides et cetra, et cetra, et cetra 6. After you have observed in detail the object to be drawn, you may begin the drawing. At first you may find the following steps helpful, so do not hesitate to refer to this section as you prepare your drawings. Biology& 100 Laboratory Manual Mr. Brumbaugh 132 Revised Summer 2016 7. Determine the size that you are going to make the drawing. The size will be determined by the size of the drawing paper (usually 8.5x11) and the shape of the object drawn. Remember that the completed drawing will have labels so be sure to leave enough space so that the finished drawing will not look crowded. The amount of magnification, or reduction, of your drawing from the actual size of the specimen is always indicated as part of the title (e.g. x1/2 or x3). 8. Determine how you are going to place the drawing on the paper. Through convention, either the anterior end (at or toward the head) or the dorsal surface (at or toward the back) is placed toward the top of the drawing. 9. Construct, by measuring the specimen with your millimeter scale and reducing or enlarging to appropriate lengths, any guide lines that you may find useful. Such guide lines should be lightly made and erased when the drawing is completed. In drawing bilaterally symmetrical structures you may find it convenient to draw a median guide line. 10. Construct outlines marking borders of the entire specimen to be drawn. At first these should be thin, light lines until you have worked out the proper form and proportions. The finished lines should be definite and continuous. Where two lines meet or cross, make them continuous, not with one or more ends showing. To show structures lying beneath other structures, use dotted lines. All light sketch lines should be erased. Fill in the details in their proper place in the outline. 11. The dedicated drawer will use drawing pencils of medium harness (3H or 4H). Softer leads will smear, and harder leads tend to cut the drawing paper. Usually do not use ink or colored pencils unless so instructed or as a final step in the preparation. Since no biological structures in nature ever have perfectly straight borders, do not use a rule to make any lines in your finished drawing. 12. In general, do not use shading. When necessary to do shading, use stippling only. Stippling involves making small dots with the tip of the pencil while holding the pencil at right angles to the paper. In stippled drawings ridges and prominences are indicated by the absence of stippling; depressions and lower parts of curved surfaces are indicated by evenly spaced dots of uniform size placed progressively closer together as the depression becomes deeper. 13. Sometimes an insert to show a close-up of a particular part of the specimen is included on the plate. This is done to show greater detail of the critter to emphasize structure function relationships or key features for identification. The same process for drawing the entire specimen should be applied to drawing the structures that appear on the insert. 14. Label the completed drawing. The labels consist of the student identification information, (name, section, date, et cetra), the plate number and title, and the names of the structures or parts illustrated. The student identification information will be placed in the upper right hand corner of the plate; the plate number and title centered at the bottom of the page; and the names of the parts placed in vertical column, parallel to each other and to the top and bottom of the page, to the right of the drawing. Solid straight lines should lead from the label to the structure. All labels should be neatly printed. 15. Make a legend that would indicate the actual size of the specimen in the drawing. this is called a scale. For example the distance between Los Angeles, CA and Seattle, WA on some maps may be only one inch but by reading the scale of the map one inch is actually about 3,000 miles. By attaching a scale to the drawing the reader is given a perspective from which to interpret the relative size of the specimen. Avoid the following common mistakes in drawing; 1. Making the drawings to diagrammatic–they should be good representations of the actual structures as seen in your specimen. Biology& 100 Laboratory Manual Mr. Brumbaugh 133 Revised Summer 2016 2. Poorly proportioned–the various parts and the whole should show the same size relationship that they have in the specimen. 3. Making the drawings too small 4. Incomplete or inaccurate labels. 5. Indefinite or “fuzzy lines.” 6. Coarse, heavy lines or uneven lines resulting from use of dull pencils or from careless work. 7. Unnecessary lines or lines without meaning. Voila, a drawing that you can treasure for the rest of your life and aid you in the successful completion of this class. YES! Will it be easy or totally rad at first. For some students yes, for others not so much. But if you stick to it things will happen, you will start to understand things about the world around you that you never noticed before. MENTAL SAFARI, WOW!! You will be doing what you came here for, learning. Biology& 100 Laboratory Manual Mr. Brumbaugh 134 Revised Summer 2016 Appendix E: How to make an Oral presentation? Oral presentations are an important means of presenting the results of your research or literature investigations to other scientists. We will use the same format that biologists use to present our literature search based papers at colleges and universities (where they are also known as “seminars") and for presentations when they attend national meetings of scientific organizations. As such, you will gain experience with a standard oral report format that you will use throughout your career as a biologist. Everyone realizes it can be uncomfortable to speak in front of a group, and it is especially hard the first time. You’ll make some mistakes—that’s part of the learning process. Please realize that any questions that you are asked by your classmates or instructor are not meant to be taken personally. So, don’t be afraid of questions and comments—they are intended to further our understanding of your literature investigation. The comments and questions made by one’s peers are important tools used by the scientific community to assist in evaluating the validity of a researcher’s experimental design, results, and conclusions. The best preparation for presentations is to understand what you did, especially why you chose the topic you research. Was there some outside influence say a family history with the topic or maybe it was some topic that peaked your interest in a news story? Whatever the reason remember that you are the “expert” in terms of our classroom, so speak confidently and calmly about your topic. Divide your presentation minutes into the following sections as you go about designing your talk: 1. Introduction This portion should be brief. Use these sentences to inform the audience in an overview about what topic or issue your paper was trying to convey. Be sure to mention why you wrote your paper and how it related to the other papers of your group. In terms of a power point this should be at most two or three slides of mainly pictures keep the text at a bare minimum otherwise we spend all our time reading and not listening. 2. Body The body should be a clear and concise display of what you found. Your information should be distilled down to the important facts and ideas about your aspect of the group’s topic. Use slides with supporting pictures to present the major trends ideas about your topic. Be sure to note whether each trend was significant or not significant. Make sure slides are easy to read and interpret, especially from a distance. These slides should be simple and straight forward . Keep each slide or group of slides with a minimum of text (that comes from YOU!) but have interesting pictures, graphs, or figures. 3. Closing Return to the question you posed in the introduction and summarize what you have just covered in your presentation. Make a transition power point slide to bridge your aspect of the topic to the next speaker. Things to Consider while Preparing for your Presentation Each person in your group must speak during the presentation. Due to time constraints your group’s presentation should last no more than 20 minutes. Plan to speak for 12 - 15 minutes so we will have 3 - 5 min. for questions and discussion with the rest of the class. Visual aids are critical to the success of your presentation. Use PowerPoint slides to present important questions, results, and conclusions. Practice your presentation a few times alone and Biology& 100 Laboratory Manual Mr. Brumbaugh 135 Revised Summer 2016 together to ensure that your presentation flows from beginning to end within the time frame. Combine each individual presentation into one seamless presentation in one file. Check with your instructor if you need special equipment for your presentation before the day your group will be presenting and not the day of your presentation . You may find it helpful to keep the following questions in mind while preparing your presentation: Do you clearly state the question you are trying to answer? Is it clear what you did to try and answer your question? Do you convey the information if a clear concise manner? Delivery of the Presentation Speak loud and clear. Project your voice to the back of the room over the ambient noise of the room. Interact with your visual aids by pointing to key features as you describe them. Try to maintain eye contact with the audience as much as possible. Remember your are the expert in the classroom about your topic. Avoid distracting behaviors, clothes, and accessories, For example, do not chew gum, lean on the podium, twirl your hair, or wear hats or distracting clothing Evaluation of the Presentation Your group’s presentation will be critiqued in two ways, by your classmates and by your instructor. Your classmates will not grade you — these comments are to help you. Each person in class will review every group by responding to the following two questions: What were the strengths of this group? What improvements could be made by this group? Classmate Evaluation When making comments about the presentation of others, keep in mind the following: Did each speaker cover the information contained in the Introduction, Body, and Closing. Was the presentation balanced among all speakers Did the presentation flow easily from speaker to speaker Were they all able to speak clearly and loud enough from the folks in the back to hear. Instructor Evaluation In addition to all of the categories above, your instructor will be interested in the following: How clearly you presented your material Whether you display understanding of what you did and why you did it, and if the data support your conclusions. You will receive an individual score. Use the rubric in your syllabus for direction. Biology& 100 Laboratory Manual Mr. Brumbaugh 136 Revised Summer 2016 Appendix F: How to search the Literature? Not all articles are created equally! The most reliable articles are from scientific journals and from the individual who conducted the study. There are 1000’s of scientific journals in the world that deal with the many fields of science. Journals publish the results of original scientific research. When scientists believe they have something of value to communicate to other scientists, they submit their work for publication. Peers that are associated with a particular society will then review it. Societies usually consist of scientists associated with universities and colleges around the world. If the research is judged to be of high quality and of value, it will be published in the society’s journal. (Note: The Audubon Society, the National Geographic Society, Wikipedia, nor the Wall Street Journal are scientific journals, reputable but not scientific!) Although much of the information in a scientific journal may be quite technical, you should be able to glean some information from it. After journals, the next best source is a popular science magazine (e.g. Scientific American, Science News, American Scientist, Discover, etc.). Since these periodicals are devoted to science, they tend to be better sources of information than general magazines such as Time or Newsweek. General popular references such as newspapers and general magazines may sometimes be helpful but don’t limit yourself to these since the information may be of unreliable quality and/or incomplete. There at several useful databases to periodicals available for your use. Some databases require the use of a computer in the Information Commons upstairs in the Holman Library (e.g. InfoTrac Health Index, an excellent database for our purposes); others are accessible from any campus computer connected to the GRCC network (e.g. ProQuest Direct). The one we will have you use is ProQuest Direct. How to find articles in ProQuest: Start Netscape Navigator or Microsoft Internet Explorer and go to Holman Library's Research Data Base Links. You should find ProQuest Direct on the list of databases (it's the second one from the top). Now click on Search ProQuest Direct. Another way to get to ProQuest: Go to the Holman Library’s home page at http://www.greenriver.edu/library/. Under “Research Tools” click on the “Databases” link. You should find ProQuest Direct on the list of databases (it's the second one from the top). Now click on Search ProQuest Direct. You are now on the Select Database screen in ProQuest. If you want to restrict your search to Peer Reviewed Articles (i.e. articles in scientific journals), then before you type in your search terms, find where it says "Peer Reviewed" on the screen, and check the box next to this. This will limit your search to Peer Reviewed articles. Type your search criteria in the search box and click the "Search" button. Don't forget to cite each of your articles correctly by following the guidelines for citing references in Appendix C. How to find books: Start Netscape Navigator or Microsoft Internet Explorer and go to Holman Library's home page at http://www.greenriver.edu/library/ Click on the "Online Catalog" link. Click on the "Basic Search" link. Type your search in the Search For: box, select "Keyword" in the Search In: box, and then click the "Search" button. Biology& 100 Laboratory Manual Mr. Brumbaugh 137 Revised Summer 2016 Unable to find a relevant book in the Holman Library, try searching at other libraries in the area by following one or more of the links at the library site. If you are not successful finding a book related to your topic in the Holman Library or the other libraries in the area try using ProQuest and restrict your search to books. Still having problems finding a book related to your topic? Try searching amazon.com. If possible, cut and paste into your Word document a short summary, description, abstract, etc. about a book related to your topic. When citing the book you found within your Word document be sure to follow the guidelines in Appendix B. Excellent Biology Web Sites—Compiled by Ken Marr, Retired from GRCC Biology Dept. http://www.google.com (One of the best search engines around!) http://www.scirus.com (One of the search engines used by scientists to seek other science works) http://www.sciam.com/ (Scientific American magazine: An extremely high quality science magazine containing articles written by experts in their field of study—One of my favorites) http://www.newscientist.com (A high quality science magazine with a biological sciences focus— Another one of my favorites!) http://www.scicentral.com/ (An excellent resource for any area of science and technology—one of my favorites—I receive weekly notices of recent papers that are of interest to me—this service is free.) http://www.sciencenews.com (A high quality science magazine with a biological sciences focus) http://ublib.buffalo.edu/libraries/units/sel/collections/ejournal2.html (Links to electronic versions of over 900 journals on the Web, covering all areas of science and technology. The content of these electronic journals varies, from full text to table of contents for the majority of journals.) http://biochemlinks.com/bclinks/bclinks.cfm (A guide with links to some of the best biological sciences and chemistry sites on the web-- including some journals and science related magazines; Includes free science related clip art and links to free clip art) http://www.nejm.org/content/index.asp (New England Journal of Medicine—one of the world’s premier medical journals) http://www.ncbi.nlm.nih.gov/Omim/ (Online Mendelian Inheritance in Man: OMIN is a database that contains summaries about every human gene so far investigated. You can obtain the official gene name, the official abbreviation, the gene map locus (where the gene is located on a certain chromosome), and information about the gene. Moreover, you can click on buttons that will give you articles in Medline (a database for medically related journals), a list of genes near the one you are interested in (a gene map), DNA sequences (DNA), and other information. Another useful site is Genbank at http://www.ncbi.nlm.nih.gov/ http://www.nlm.nih.gov/ (Medline: A database of the National Library of Medicine, part of the National Institutes of Health (NIH). This the largest collection of medical information in the world, containing more than 9 million references from medical journals from all over the world. http://cancer.med.upenn.edu/ (Oncolink: the first of its kind on the Internet—an excellent site that disseminates cutting edge information relevant to the field of oncology (cancer research). Aims to educate health care personnel, patients, and other interested parties.) http://www.quackwatch.com/ (“A Guide to Health Fraud, Quackery, and Intelligent Decisions;” An interesting site that helps one to distinguish between legitimate healthcare treatments and quackery—The physician responsible for this site has written many books and scientific papers over the years. His ideas are very mainstream—perhaps too mainstream? Some of the views expressed may not be totally objective. At times he has quite harsh comments concerning “alternative medicine.”) Biology& 100 Laboratory Manual Mr. Brumbaugh 138 Revised Summer 2016 http://www.audubon.org/ (Audubon is a high quality magazine that deals with environmental issues and wildlife conservation) http://www.biomednet.com/hmsbeagle (This is one of my favorites—A weekly publication that covers many of the more important advances in the biological sciences. Requires membership— which is free as is an email subscription) home page of the H.M.S. beagle: http://www.biomednet.com/home http://genetics.nature.com/ (a journal produced by Nature…Gives you access to the contents, but you must pay to see the text of the articles—Available for free in the libraries of most research universities) http://www.nature.com/ (Nature is a very prestigious scientific journal. This site gives you access to the contents. Although some parts of the site are free, you must pay to see the text of the articles— but they are available for free in the libraries of most research universities) http://flybase.bio.indiana.edu/ (FlyBase: a comprehensive searchable database for information on the genetics and molecular biology of Drosophila—the fruit fly) http://www.exploratorium.edu/exhibits/mutant_flies/mutant_flies.html (Has pictures and descriptions of mutant fruit flies) Biology& 100 Laboratory Manual Mr. Brumbaugh 139 Revised Summer 2016 Appendix G: Classification of the Animal & Plant Kingdoms Kingdom/Phylum Highlights Kingdom Animalia 1. General Characteristics a. Animals show a variance in form and structure but hold some characteristics in common such as mobility, dedicated to sexual reproduction, heterotrophic, and multi-cellular. 2. Major Taxa a. Phylum Porifera (Pore Bearer) - the sponges (@ 9,000 sp) Complexity Level: Cellular level of Organization (Intracellular digestion). No tissues or organs. No head, mouth, gut, nervous system, sense organs. The blob that doesn't seem to care. Filter feeders (choanocytes and pores). On making waves, or at least drawing a current. Having only one opening makes for problems. (Osculum). Asymmetry and/or Radial symmetry (like the spokes of a wheel) - or on living without a head. Body wall made up of spongin + spicules. (75% produce chemical toxins in walls) b. Phylum Cnidaria - animals with stinging cells. (@9,000 sp) Complexity Level: Tissue level of Organization (Extracellular digestion) Getting organized. Tissue comprised of groups of specialized cells: Nerve cells for coordination, Muscle cells for getting around and hauling in food, Gut cells for working over the food, and, Nematocysts (stinging cells). Mesoglea sandwich - Two germ layers. Only two layers of cells - or having only an inside and outside. (Ectoderm and Endoderm) Blind sac body plan - gastrovascular cavity. Having only one opening makes for problems. Radial symmetry (like the spokes of a wheel) - or on living without a head. Polymorphism: medusa and polyp body types. Some different kinds: Sea anemones, Hydra, Colonial Hydrozoans, Jellyfish. c. Phylum Ctenophora - comb jellies, sea gooseberries, or sea walnuts Complexity Level: Between cellular and tissue. Active feeders with a soft body. Shaped like a cup or upside down bell with eight rows of cilia beating from the base to the cup opening. Bi-radial symmetry. Only one intake and output opening Hermaphroditic d. Phylum Platyhelminthes - the flat worms. (@20,000 sp) Complexity Level: Organ systems Definite polarity and Bilateral symmetry. Biology& 100 Laboratory Manual Mr. Brumbaugh 140 Revised Summer 2016 e. f. g. h. Three germ layers - add mesoderm. More than two layers of cells makes possible some definite organs, not mere tissues, and other big deals. But alas, the food gathering apparatus still has only one opening. (Tubular pharynx). No body cavity around the gut - Acoelomate. Flat, generally thin, unsegmented. Some different kinds: Planaria, leafworms, flukes, and tapeworms. Phylum Nemertea - the ribbon worms Complexity Level: Organ systems Definite polarity and Bilateral symmetry. Tube within a tube body plan. One way traffic - or a mouth, a gut, and an anus forming a complete system, but no body cavity around the gut Acoelomate. One way traffic - or a mouth, a gut, and an anus forming a complete system. Closed circulatory system, most primitive group with a circulatory system. Special body cavity (rhynchocoel) which contains a protractible proboscis. Phylum Nematoda - round worms (>12,000 and maybe several more) Complexity Level: Organ System. Groups of organs now working together in a system - the ultimate in design. Tube within a tube body plan. One way traffic - or a mouth, a gut, and an anus forming a complete system. Pseudocoelomate - fluid filled body cavity between the digestive tract and the body wall - derived from blastocoel (fluid filled cavity of blastula). Not lined with peritoneum. Bilateral symmetry. Numerous modifications of the animal way of life are illustrated by a variety of phyla at about the same level of development as the round worms. Phylum Nematomorpha - horsehair worms Phylum Acanthocephala - spiny-headed worms Phylum Gastrotricha - spiny bodied worms Phylum Rotifera - wheel worms Phylum Bryozoa - moss animals Phylum Brachiopoda - lamp shells Phylum Phoronida - tentacle worms Phylum Chaetognatha - arrow worms Phylum Sipunculoidea - peanut worms Phylum Mollusca - of shells and feet, and things like that and a mantle to cover their guts (>100,000 sp) (@ 60,000 fossil sp) Complexity Level: Organ System. (Protostomes). Body - Soft with true coelom (Eucoelomate). Plan - Muscular Foot, Viscera, and Mantle. (Mantle = specialized dorsal body wall which covers the internal organs and secretes the shell) Open circulatory system w/hemocoel. Bilateral symmetry or secondarily asymmetrical. Biology& 100 Laboratory Manual Mr. Brumbaugh 141 Revised Summer 2016 i. j. k. l. Unsegmented and many with a shell. Some different kinds: Class Polyplacophora (Amphineura) - chitons. Eight plates of armor. Class Bivalvia - Two shells and a foot for digging. Class Gastropoda (meaning stomach foot). On sliding around on your belly. Class Scaphopoda - tooth shells. Class Cephalopoda (meaning head foot). Tentacles and monsters of the deep. Phylum Annelida - segmented worms. Tidy little compartments. (15,000 sp) Complexity Level: Organ-system level of development is now clearly established. (Protostomes) Beginning of segmentation, an event which allows various regions of the body to specialize. Segmented: body is arranged in a repetitive linear sequence of similar parts. Also called metamerism (each segment called a metamere or somite) May be external and/or internal. Segments divided externally by grooves or serial repetition of appendages. Eucoelomate. Setae - small external bristles (except leeches). An indication of appendages (arms and legs). Bilateral symmetry. Some different kinds: Earthworms, Clamworms, Leeches. Phylum Tardigrada - little water bear. Phylum Onychophora Peripatus. The missing link? Phylum Arthropoda - animals with jointed legs. Being most popular. (1 million sp. described > 50 million more "unknown") Complexity Level: Organ-system level (Protostomes) Segmented - Jointed appendages (specialized body regions called Tagma = Head, thorax, abdomen) Eucoelomate. Exoskeleton (chitinous). Bilateral symmetry. Why so successful? Highly specialized segmentation. Extreme polarity, allowing for such advanced coordination as flight. Striated (rather than smooth) muscle, allowing for rapid movement. Jointed appendages showing all sorts of special structural adaptations. All systems now present, including a skeletal system. Primitive intelligence and social instincts in some. Tremendous rates of reproduction. Some different kinds: Class Diplopoda - millipedes. Thousand legged critters. Class Chilopoda - centipedes. Class Crustacea - five pair of walking legs (crabs, shrimp, pill bugs) Biology& 100 Laboratory Manual Mr. Brumbaugh 142 Revised Summer 2016 Class Arachnida - spiders, etc. Four pair of walking legs. Class Insecta - three pair of legs and sometimes wings too (insects) m. Phylum Echinodermata - Spiny-skinned animals. (7,000 sp.) On the road to back-boned animals. Complexity Level: Organ System. (Deuterostomes). A unique water hydraulic system - the water vascular system. Hydraulic Tube feet. Some with unique hydraulic pincers - pedicellaria. Endoskeleton of calcareous plates. Adult radial symmetry (like the spokes of a wheel) - or on living without a head. Unsegmented with Oral and Aboral sides. Eucoelomate. Some different kinds: Class Crinoidea - Sea lily and Feather Star. Class Asteroidea - Sea stars. Class Ophiuroidea - Brittle stars. Class Echinoidea - sand dollar, sea urchin, and heart urchin. Class Holothuroidea - Sea cucumbers. n. Phylum Urochordata - sea squirts. They lose their backbone. Our distant relatives. o. Phylum Chordata - back-boned animals. (50,000 sp) Complexity Level: Organ System (Deuterostomes). Eucoelomate. Metamerism. (Somites of backbone). Highly cephalized. Bilateral symmetry. Unique shared characters and different kinds Dorsal notochord (flexible, rod-like structure that provides skeletal support made of cartilage. Extends to tail, point of attachment for body wall musculature). Single hollow dorsal nerve cord. Postanal tail. Myotomes (muscle bundles) are segmentally arranged in a non-segmented trunk. Some different kinds: Class Agnatha - jawless fish - lampreys and hagfish. Class Chondrichthyes - cartilaginous fish. Life without a bony skeleton sharks and rays. Class Osteichthyes - bony fish. A hard skeleton a last. Class Amphibia - amphibians. Life in the water and on the land. Class Reptilia - reptiles. Spending life on the land. Class Aves - Birds. Class Mammalia - mammals. That's our crowd, gang. Biology& 100 Laboratory Manual Mr. Brumbaugh 143 Revised Summer 2016 a. Subclass Prototheria - monotremes - Duck billed platypus and echidna. b. Subclass Metatheria - marsupials - wallabies, wombats, kangaroos, opossums, koalas. c. Subclass Eutheria - placentals - walrus, whales, Fido, Felix, King Kong, You. Kingdom Plantae Botany and Biology texts treat the classification of the plant kingdom in different ways. For example, the term Division is used in botany to denote the Phylum level of classification. Biology texts tend to use the term Phylum instead of Division when referring to this level of classification in plants. Most texts break the plant kingdom into two groups: vascular and non-vascular plants. The non-vascular plants are termed Bryophytes. The Bryophytes include the groups of plants known as mosses, liverworts, and hornworts (@23,000 sp). They have the following general characteristics in common: seedless, spore producing, nonvascular, and haploid dominance. The vascular plants are termed Tracheophytes. Included in this group are the very simple horsetails, the ferns, large conifers, and the flowering plants (@350,000 sp). They have a wide range of characteristics except for each having vascular tubing cells called xylem (to move water and minerals from the roots) and phloem (to move sugars from the leaves). 1. Division Bryophyta - Mosses a. Life style of alternation of generation (alternates between sporophyte (makes spores that are diploid) and gametophyte (makes gametes that are haploid)) b. Pigments of chlorophyll (green). c. Matting shape on rocks, tree, driveway, etc., sporophytes penetrate through gametophyte matting. d. No roots get moisture and minerals from the air. Like moist climates. e. Leaflets on gametophyte 2. Division Hepatophyta - Liverworts a. Life style of alternation of generation b. Pigments of chlorophyll c. Grows in shaded moist areas d. Gametophyte looks like a clump of small leaves with the sporophyte structure growing through with a small brown knob containing the spores e. Rudimentary vascular like cells 3. Division Anthocerophyta - Hornworts a. Life style of alternation of generation b. Pigments of chlorophyll some have xanthophylls (reds) c. Grows in shaded moist areas d. Gametophyte looks like a clump of small leaves with the sporophyte structure growing through in a hollow tube like structure e. Rudimentary vascular like cells 4. Division Sphenophyta - Horsetails a. Seedless, spore producing Biology& 100 Laboratory Manual Mr. Brumbaugh 144 Revised Summer 2016 b. Vascular tubes of xylem (water and minerals) and phloem (sugars) c. Some sporophyte stems photosynthetic, others non-photosynthetic d. Pigments of chlorophyll e. Grow in areas with shade and more toxic soils 5. Division Pteridophyta - Ferns (@ 12,000 sp) a. Seedless, spore producing. Largest group of seedless vascular plants b. Vascular c. Pigments chlorophyll d. Mainly tropical and temperate habitats e. Some sporophyte stems photosynthetic, others non-photosynthetic 6. Division Coniferophyta - Conifers a. Seed-bearing b. Vascular c. Pigments of chlorophyll d. Cone bearing e. Needle-like or scale-like leaves (microphylls) 7. Division Anthophyta - Flowering plants (@ 250,000 sp). a. Seed-bearing. Largest, most diverse group of vascular seed-bearing plants b. Vascular with megaphyll leaves c. Pigments of chlorophyll, carotenoid (orange), and xanthophylls d. Only organisms that produce flower and fruits e. Some different kinds: Class Dicotyledonae - dicots: rose, maples, cacti, lettuces, beans, cotton, elms, blackberry, most trees and shrubs other than conifers and others. Class Monocotyledonae - monocots: palm trees, lilies, orchids, bamboo, wheat, corn, pineapples, grasses, sugar cane and others Characteristics of Flowering Plants Classes # of seed leaves Floral parts Leaf Vein Array Vascular Bundles Root Stystem Biology& 100 Laboratory Manual Mr. Brumbaugh Monocotyledon One In threes Parallel Veins Random array Pattern Fibrous 145 Dicotyledon Two In fours or fives Net Veins Ring array Pattern Taproot Revised Summer 2016