Name Period Honors Biology Lab Manual Mt. Lebanon Biology Department Revised: April 14, 2011 Page |1 Table of Contents 2……………………………………………………………………….... Laboratory Safety 4…………………………………………………………………….Lab Report Guidelines First Semester 5……………………………………………………..What are the constraints on cell size? 8……....What are the similarities and differences between cells from different kingdoms? 13…………………………………………………………………………….Is yeast alive? 15…………………………...Identifying the organic compounds in different food sources 19…………What are the effects of different wavelengths of light on photosynthetic rate? 21…………………………………………………………...Respiration of sugars by yeast 25…………………………………………………….…………………Making Root Beer 27……………………………………………………….…..Case Studies – Trophic Levels 29……………………………………………..How do cell membranes regulate transport? 32…………………..………What environmental factors affect xylem transport in plants? 34…………………………What are the effects of exercise on CO2 output and heart rate? 36……………………………………………………What can you learn by testing urine? 41………….………Which antibiotic or disinfectant is best at controlling E. coli growth? 43……...…………………How do germinating seeds respond to environmental changes? 44……...………………………………………..……………………….Sensory Receptors 49…………………………................................…………….Testing The Nervous System 50……………………………………………….....…..Investigating Invertebrate Behavior Second Semester 54……………………………………………………………..…..Banana DNA Extraction 56………...During the cell cycle, which stage do onion root cells spend the most time in? 59…...……………………………….......…………………………..Principles of Genetics 66…………………………………………………………...Applying Mendelian Genetics 71……….…..How can blood typing be used to determine parent-offspring relationships? 78……...............................................................................................................Karyotyping 80………………………………………………………………….……Radioactive Decay 83...……….....……………………………………………….Examining the Fossil Record 89………………………...….Natural Selection: The driving force of evolutionary change 101……….……………………………………...……Animal Classification and Diversity 102………………………………………….……………………….……Squid Dissection 105………………………………………………………………...……….Frog Dissection Page |2 Laboratory Safety Purpose: One of the first things a scientist learns is that working in the laboratory can be an exciting experience. But the laboratory can also be quite dangerous if proper safety rules are not followed at all times. To prepare you for a safe year in the laboratory, read over the following safety rules then read them a second time. Make sure you understand each rule. If you do not, ask your teacher to explain any rules you are unsure of. Pre-Lab Rules 1. Come prepared to work. Be seated and turn in any items due at the beginning of the period. Be sure that you understand the procedure (after the introduction) and the possible hazards associated with it. 2. Get and apron or goggles if instructed and check the board for the lab pages to access. 3. Keep your laboratory area clean and free of unnecessary books, papers, and equipment. Place any food, drink, books, book bags, etc, under the desk. 4. Keep the aisles clear. Push your chair under the desk when not in use. 5. Be serious and alert when working in the laboratory. Never "horse around" in the laboratory. 6. Read all directions for an investigation several times. Follow the directions exactly as they are written. If you are in doubt about any part of the investigation, ask your teacher for assistance. 7. Never perform activities that are not authorized by your teacher. Obtain permission before "experimenting on your own." 8. Never handle any equipment unless you have specific permission. 9. Never eat or taste anything in the laboratory unless directed to do so. This includes food, drinks, candy, and gum, as well as chemicals. Dress Code 1. Remove or tie back any article of baggy or loose clothing or jewelry that can hang down and touch chemicals and flames. 2. Long pants are also recommended, but not required in the lab. 3. It is recommended that jewelry not be worn in the lab. 4. Many materials in the laboratory can cause eye injury. To protect yourself from possible injury, wear safety goggles whenever you are working with chemicals, burners, or any substance that might get into your eyes. 5. Wear a laboratory apron or coat whenever you are working with chemicals or heated substances. 6. Tie back long hair to keep your hair away from any chemicals, burners and candles, or other laboratory equipment. 7. Do not wear sandals or open toed shoes in the laboratory. Never walk around the laboratory barefoot or in stocking feet. First Aid & Medical Information 1. Become aware of the location of the first aid kit, safety shower, and eye wash station. But remember that your teacher should administer any required first aid due to injury. Or your teacher may send you to the school nurse or call 911. 2. Report all accidents, no matter how minor, to your teacher immediately. 3. Know how to call the principal, nurse, and 911 in case of an emergency. 4. After the introduction to the lab exercise, notify your instructor of any medical concerns, allergies, or asthma problems that are applicable that investigation. 5. It is recommended that contact lenses not be worn in the laboratory 6. Learn what to do in case of specific accidents such as getting acid in your eyes or on your skin. (Rinse acids off your body with lots of water.) Fire Safety 1. Maintain a clean work area and keep all materials away from flames. 2. Know where and how to report a fire. Find out the location of the fire extinguisher, fire alarm, fire blanket, fire exit route, and phone. If an emergency occurs, know how to call 911, the principal, or nurse. Report any fires to your teacher at once. 3. Never use a heat source such as a candle, burner, or hot plate without wearing safety goggles. 4. Look for the Kimex or Pyrex labels before heating glassware. This label indicates that the glassware is safe at high temperatures. 5. Never heat a chemical you are not instructed to heat. A chemical that is harmless when cool can be dangerous when heated. 6. Never heat liquid in a closed container. The expanding gasses produced may blow the container apart, injuring you and others. Page |3 7. Point a test tube or glassware that is being heated away from you and others. Chemicals can splash or boil out of heated glassware. 8. Never reach across a flame. 9. Make sure you know how to light a Bunsen burner and turn on the gas jets. Your teacher will demonstrate the proper procedure for lighting a Bunsen burner when that procedure is encountered in lab. If the flame leaps out of a burner toward you, turn the gas off immediately. Do not touch the burner. It may be hot. Never leave a Bunsen burner, hot plate, or open flame unattended. Someone needs to be watching it at all times. 10. Never pick up a container that has been heated without first holding the back of your hand near it. If you can feel the heat on the back of your hand, the container may be too hot to handle. Use a clamp, tongs, or heat-resistant gloves when handling hot containers. Using Chemicals Safely 1. Keep your hands away from your face, eyes, mouth, and body while using chemicals or preserved specimens. 2. Never mix chemicals for the "fun of it." You might produce a dangerous, possibly explosive substance. 3. Never touch, taste, or smell a chemical that you do not know for a fact is harmless. Many chemicals are poisonous. If you are instructed to note the fumes in an investigation, gently wave your hand over the opening of a container and direct the fumes toward your nose. Do not inhale the fumes directly from the container. 4. Use only those chemicals needed in the investigation. Keep all lids closed when a chemical is not being used. 5. Never use the same transfer devices (i.e., pipettes, test tubes) for different chemicals unless they are washed thoroughly before the next transfer. 6. Take extreme care not to spill any material in the laboratory. If spills occur, ask your teacher immediately about the proper cleanup procedure. Never simply pour chemicals or other substances into the sink or trash container. 7. Dispose of all chemicals as instructed by your teacher. To avoid contamination, never return chemicals to their original containers. 8. Solid chemicals and metals are to be disposed of in the proper waste containers, not in the sink. Check the label of all waste containers twice before adding your chemical waste to it. 9. Be extra careful when working with acids or bases. Pour such chemicals over the sink, not over your work bench or notebooks. 10. When diluting an acid, pour the acid into water. Never pour the water into the acid. 11. Rinse any acids off your skin or clothing with water. Immediately notify your teacher of any acid spill. Glassware 1. Never eat or drink from laboratory glassware. Clean glassware both before and after each investigation. 2. Never force glass tubing into a glass stopper. This may cause the glassware to break injuring you and possibly others. Your teacher will demonstrate the proper way to insert tubing into a stopper when encountered in lab. 3. Keep in mind that hot glassware will not appear hot. Never pick up glassware without first checking to see if it is hot. Use the proper equipment for handling hot glassware, i.e., test tube holders, heat resistant gloves, etc. 4. Report all broken glassware to your teacher immediately. Dispose of the glassware in the proper container. Sharp Instruments 1. Handle scalpels, scissors, razor blades, probes, and all sharp equipment with extreme care. Never cut material toward you; cut away from you. 2. Notify your teacher immediately if you cut yourself or receive a cut. 3. Remember that scalpels, razor blades, probes, etc, are laboratory tools, but are considered weapons when seen outside of the laboratory classroom. Living Organisms 1. No investigations that will cause pain, discomfort, or harm to mammals, birds, reptiles, fishes, and amphibians should be done in the classroom or at home. 2. Treat all living things with care and respect. Do not handle any organism in the laboratory unless given permission to do so. 3. Your teacher will instruct you on the proper handling of each species brought into the classroom. 4. Treat all microorganisms as if they were harmful. Use antiseptic procedures, as directed by your teacher, when working with microbes and bacteria. Dispose of all bacterial specimens as directed by your teacher. 5. Be sure to wash your hands after handling any organisms in lab. End of Lab Rules 1. With 5 minutes remaining in the period, clean up your work area and return all equipment to its proper place. 2. Wash hands, lab benches, and sink areas after every investigation. 3. Use specified antiseptic techniques if bacterial microbes were used in the lab. 4. Turn off all burners and faucets before leaving the laboratory. Check that the gas line leading to the burner is off as well. Page |4 Lab Report Guidelines Title Describes lab content concisely, adequately, appropriately Objectives Includes the question to be answered by the lab Successfully establishes the scientific concept of the lab Effectively presents the objectives and purpose of the lab Hypotheses Includes both null and alternative hypothesis Hypothesis is based on research and/or sound reasoning Hypothesis is testable. States hypothesis and provides logical reasoning for it Methods Includes materials and summary of procedure Gives enough details to allow for replication of procedure Results Includes data tables, visual representations of data, and statistical analysis Presents visuals clearly and accurately Presents verbal findings clearly and with sufficient support Results and data are clearly recorded, organized so it is easy for the reader to see trends. All appropriate labels are included No interpretation Discussion Interpretations: Discuss what the trends in your data indicate. Discuss trends related to the question at hand as well as any interesting unrelated trends you may have seen that are not directly related to the question at hand. Conclusion: State whether your hypothesis was supported or refuted and how confident you are in this claim. If your results were inconclusive, discuss why. Develop reasoning as to why results show what they do. Discuss the theory behind your results. Mistakes: These could be flaws in your design and/or mistakes in your execution, both of which can be fixed by redesigning or repeating the experiment. If you were unable to fix your mistakes during the experiment, mention them here. Error: Sources of error are factors that lower the quality of your data that are more or less out of your control. No data is perfect; all experiments have sources of error. Suggestions: If your experiment was inconclusive, suggest ways to redesign or repeat the experiment in order to make it conclusive. If your results were conclusive (hypothesis was either supported or refuted) but not extremely confident, suggest ways to improve this confidence. If your conclusion was sufficiently confident, suggest a new experiment by asking a follow-up question. Also, suggest any new hypotheses that might arise from any interesting trends you noticed that were unrelated to the initial hypothesis. Applications: Discuss how your conclusions to this lab relate to or can be applied to real life situations. Discuss why the knowledge gained from this lab is useful. Page |5 What are the constraints on cell size? Objective Determine how different cell sizes affect nutrient intake and output. Materials list (per group) Each class will have four students. Within each group, two students will run experimental setup 1 and two students will run experimental setup 2. Experimental Group I Plastic Spoon 250 ml beaker 3 Agar-Phenolphthalein blocks (1 cm, 2cm, and 3cm sizes) 100 ml of 0.4% sodium hydroxide solution Paper towel Scalpel Metric ruler Experimental Group II Plastic Spoon 3 Agar-Sodium chloride blocks (1 cm, 2cm, and 3cm sizes) 1-600 ml beaker Distilled water bottle Paper towel Scalpel Metric ruler Ring Stand Utility clamp Vernier probe for conductivity Laptop plus Vernier interface equipment Power cord, USB cord, Lab Pro Materials list (per class) 12 plastic spoons 6 ice cubed shaped agar-phenolphthalein blocks 6 ice cubed shaped agar-sodium chloride blocks 600mL of 0.4% sodium hydroxide solution 6 distilled water bottles 6-250mL beakers 6-600mL beakers 12 metric rulers 12 scalpels 6 ring stands 6 utility clamps 6 Vernier setups including laptops and conductivity probes Page |6 Procedure – Experimental Group I: Testing Sodium Hydroxide Transport 1. Using the plastic spoon, place the 3 agar-phenolphthalein blocks in the beaker. Pour 100 mL of the 0.4% sodium hydroxide solution over the blocks. Allow the blocks to remain in the solution undisturbed for 10 minutes. 2. After 10 minutes, remove the blocks with the plastic spoon. Place them on the paper towel and blot them dry. 3. Cut each block in half with the scalpel. Using the metric ruler, measure the distance in millimeters that the pink color has diffused into each block. Record this measurement in the appropriate data table. Procedure – Experimental Group II: Testing Sodium Chloride Transport 1. Set up the utility clamp, Conductivity Probe, and ring stand as shown. 2. Connect the Conductivity Probe to the computer interface. Prepare the computer for data collection by opening the file “02 Limits on Cell Size” from the Biology with Vernier folder of Logger Pro. 3. Set the selector switch on the side of the Conductivity Probe to the 0 – 20000 µS/cm range. 4. Pour 300mL of tap water into the 600mL beaker. Position the Conductivity Probe in the water so the tip is about 2 cm from the bottom of the beaker. 5. Place the solid agar cube sample in the beaker and begin data collection by clicking . Stir the water using the stirring rod. Data collection will automatically end after two minutes. 6. Determine the rate of ion exchange by performing a linear regression of the data: a. Click the Linear Fit button, , to perform a linear regression. A floating box will appear with the formula for a best fit line. b. Record the slope of the line, m, as the rate in the appropriate data table.. 7. Empty the water from the beaker and rinse it thoroughly. Rinse the probe with clean, distilled water. Blot the outside of the probe tip dry with a tissue or paper towel. It is not necessary to dry the inside of the hole near the probe end. 8. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu. 9. Repeat Steps 4 – 8 for the 2cm agar cube. 10. Repeat Steps 4 – 8 for the 3cm agar cube. Page |7 Data Tables and Calculations Agar Blocks - Calculations Cube Size Surface Area Volume Surface Area-to-Volume (cm2) (cm3) Ratio 1 cm 2 cm 3 cm Surface area = number of surfaces x length x width Volume = length x width x height Surface area-to-volume ratio should be listed with a colon (ie. 3:1 or 5:1, etc). Data - Experimental Group I: Testing Sodium Hydroxide Transport Cube Size Distance traveled by Ratio of distance traveled- Most efficient in intake of sodium hydroxide to-volume chemicals 1 cm 2 cm 3 cm The most efficient cube size will be the one which has the highest ratio of distance traveled to volume Data - Experimental Group II: Testing Sodium Chloride Transport Cube Size Rate (m) Ratio of conductivity-to- Most efficient in export of (µS/cm/s) volume chemicals 1 cm 2 cm 3 cm The most efficient cube should be the one which results in the highest conductivity-to-volume ratio Discussion 1. How did the experiments using sodium hydroxide and sodium chloride model intake and output in cells? 2. Define efficiency in terms of cell intake and output. How does this lab measure efficiency? 3. What aspect of cells that we measured determines their ability take in and release materials? 4. What is the relationship between efficiency and cell size? What evidence do you have? 5. What are the constraints on cell size? What is the smallest a cell can be? Why? What is the largest a cell can be? Why? Page |8 What are the similarities and differences between cells from different kingdoms? Introduction Ever since the first microscope was used, biologists have been interested in studying the cellular organization of all living things. After hundreds of years of observations by many biologists, the cell theory was developed. The cell theory states that the cell is the structural and functional unit of living things. Cells contain structures called organelles that carry out life processes. In cells, similarities and differences exist because of varied life functions. There are two major divisions into which all cells fall – prokaryotic and eukaryotic. Prokaryotic cells are cells the lack a nucleus and membrane-bound organelles. Bacteria and related microorganisms are prokaryotes. Eukaryotic cells are cells that contain a nucleus and membrane-bound organelles. Organisms such as animals, plants, fungi, and protists are all eukaryotic. Objectives Observe cells on various slides to examine the differences between prokaryotic and eukaryotic cells and plant and animal cells Compare and contrast the structures of different cells Materials (per group) Microscope Medicine dropper Rulers Glass slide Coverslip Methylene blue stain Lens paper Toothpicks Forceps Water Elodea leaf Prepared slides of prokaryotic and eukaryotic cells Pre-Lab Part I: Parts of the Compound Light Microscope Use the diagram of the microscope to familiarize yourself with the various parts and their functions. Page |9 Pre-Lab Part II: Magnification of the Lenses It is often difficult to know the actual size of an object being observed. Magnification causes us to lose the idea of actual size. You cannot hold up a ruler to a paramecium or a plant cell while it is under the microscope. There size must be measured indirectly – that is, it must be compared to the size of something you already know. The diameter of the microscope field seen through the eyepiece is a convenient standard to use. To measure objects under a microscope, a unit called micrometers (μm) is used. Complete the following data table. To determine the total magnification, multiply the magnification of each objective by the magnification of the eyepiece. Objective Magnification of Magnification of Total Objective Eyepiece Magnification Low-power Medium-power High-Power (Oil) Part I: Measuring with the Compound Light Microscope Procedure Microscopic measurements are frequently of great importance to the biologist. The smallest unit of macroscopic metric measurement is one millimeter (mm), or about 1/25 in. However, this is an extremely large unit for microscopic measurement. For measuring microscopic materials, the biologist uses a unit known as a micron (.u), which is 0.001 mm. There are 1000 um in 1 mm. Research microscopes are often equipped with an optical micrometer, a glass disk with a micron scale, which is placed in the ocular, as shown. By moving the slide, an object may be placed along the scale and measured accurately. Without an ocular micrometer, however, you can determine the approximate size of many specimens by comparing them with the known diameter of the low-power and high- power fields. Using a metric ruler, determine the diameter for the field of view at low, medium, and high power. Record your observations. Low Power Medium Power High Power ____ ____ ____ um um um P a g e | 10 Part II: Observing and Comparing Cells Procedure 1. Using appropriate technique, obtain a microscope and prepare it for use. 2. Obtain various prepared slides from your teacher for observation: two types of bacteria, one protist, and one fungus. Locate each of the cell types under the microscope. a. In the data table, write the name of each type of cell (ex. E. coli) that you examined. Describe the general shape and estimate the size of the cell in the spaces provided. Be sure to determine what power magnification you are best able to observe the cells under in order to accurately estimate their sizes. b. At high power, look for structures unobservable at low power. Put a check mark next to the cell structures that you are able to observe. These are difficult. Do your best. c. Based on your observations, decide if the cell is prokaryotic or eukaryotic and record this in the data table. d. In the appropriate place in the observation section, draw and label what you see under the microscope in color. Be sure to record the magnification of the microscope. 3. Examining Plant Cells a. Place a drop of water in the center of a clean glass slide. b. With forceps, remove a leaf from the Elodea or Java Moss plant and place it on the drop of water on the slide. Make sure that the leaf is flat. c. Carefully place a coverslip over the drop of water and leaf. d. Once the slide has been prepared, repeat step number 2. 4. Examining Animal Cells a. Place a drop of water in the center of a clean glass slide. b. Using the flat end of the toothpick, gently scrape the inside of your cheek. Do not draw blood. Cells should be stuck to your toothpick although it may appear to be just saliva. c. Stir the water on the slide with the end of the toothpick to mix the cheek cells with the water. Dispose of the toothpick as instructed by your teacher. d. Put one drop of methylene blue stain on top of the drop of water containing the cheek cells. Be careful not to stain your hands and clothing. e. Wait one minute, then carefully place a coverslip over the stained cheek cells. f. You may remove excess stain from under the coverslip by placing a piece of paper towel under the edge of the coverslip. The stain will be absorbed by the paper towel. Dispose of the paper towel after use. g. Once the slide has been prepared, repeat step number 2. 5. Clean and dry all of the slides and coverslips. 6. Return microscopes to your storage areas. P a g e | 11 Data Cell Structures Nuclear Envelope Cell membrane Size Prokaryotic or Cell Type Shape Cytoplasm (um) Eukaryotic Vacuoles Cell wall Nucleus Plastids Observations Slide 1 – Bacteria Mag. Slide 2 – Other Mag. Cell Type Cell Type Slide 3 – Protista Mag. Slide 4 – Fungi Mag. Cell Type Cell Type P a g e | 12 Slide 5 – Plant Mag. Slide 6 – Animal Mag. Cell Type Cell Type Cheek Epithelial Cells Discussion 1. Based on your observations, do all cells have the same shape and size? Provide examples to support your answer. Explain why or why not. 2. What structures are common to all cells? What structures are specific to certain cells? Elaborate for each. 3. Why are stains such as methylene blue used when observing cells under the microscope? 4. What is the advantage of using a wet-mount preparation instead of a dry mound preparation in the study of living cells? 5. In general, the surface of a tree has a harder “feel” than does the surface of a dog. What cell characteristic of each organism can be used to explain this difference? 6. Classify the following unknown cells. Be as specific as possible, but if more than one group of organisms applies, list all possibilities. a. Cell wall, nucleus, mitochondria – 30 um. b. Rod shaped, cell wall – 2 um. c. Cell membrane, no cell wall, nucleus, ribosomes – 45um. P a g e | 13 Is yeast alive? Objective To find out whether yeast is alive, we first need to think about what makes something alive. There are several characteristics of living things. All living things must: (1) be comprised of cells, (2) must reproduce, (3) use energy, (4) respond to their environment, and (5) grow and develop. We will use these characteristics to determine if yeast is alive. Materials 4 – 16 x 150mm test tubes Test tube racks Plastic droppers Glass slides Cover slips Lens paper Flour Salt Baking Soda Sugar Distilled water Dry yeast packets Metric rulers Microspoons Digital Microscopes Scotch Tape Petri Dishes w/ YED Agar Markers Digital Camera 7” Balloons – 4/group Procedures I. Do yeast contain cells? 1. Create a wet mount slide of yeast and observe it under the microscope. Draw your observations or create a digital micrograph. These should be captured in high power. If using digital microscopes, include your name(s) and magnification on the digital image before it is captured. Include your images in your report. II. Do yeast grow, develop, and reproduce? 1. Obtain an agar plate containing yeast media. Label the bottom of the dish with your name, teacher, and class period. 2. Add 5 drops of the warm yeast solution to the plate. 3. The plates should be photographed with a digital camera on top of a white sheet of paper that has your name(s), teacher, and period. Be sure to note that this is your initial observation. 4. Your plates will be incubated at 37° C until the next class. (Formula: F=9/5C +32) 5. After at least one day, inspect your plate. Take another digital picture of your plate after the incubation period using the same methods as before. Be sure to note that this is your final observation. Compare your two digital photographs. Both, the before and after incubation photos should be included in your report. P a g e | 14 III. Do yeast utilize energy and respond to their environment? Organisms that obtain energy from organic food sources give of gases, such as CO2, as a waste product. We will carry out a test for yeast metabolism in different environments. In other words, we will be indirectly testing whether yeast can utilize sugar as a food source. Then we will test if there are differences in metabolism under different situations as a response. 1. Set up four test tubes in a test tube rack. 2. Label each tube with a number, 1-4, and group name. All test tubes will contain yeast and water (3/4 of the tube should be filled with warm yeast solution). These amounts should be consistent in each tube. Test tubes 2-4 will also contain sugar. Test tubes 3 and 4 will also have either salt, flour, or baking soda (you choose). What amounts of each variable should be added to the tubes? 3. Hold your thumb over the test tubes and shake them vigorously to thoroughly mix the contents. Cover the opening of each test tube with a balloon to catch any gas that is formed. Tape the balloon in place so it cannot be pulled off. 4. Allow the tubes to sit overnight at room temperature. Record the diameter of the balloon the next day in the table below. This table should be included in your report. TEST TUBES BALLOON DIAMETER (All tubes contain yeast and water) Over 24 hours Test tube 1: Control – Yeast solution only Test tube 2: Sugar Test tube 3: Sugar and Environmental Condition A Test tube 4: Sugar and Environmental Condition B In your lab report, be sure to include the following items. Results Part I: 1 Digital micrograph picture or drawing Part II: 2 Digital pictures (initial and final) Part III: Balloon Diameter Data Table Discussion 1. Describe your observations for each part of the experiment. 2. Explain whether your data confirms if yeast is alive. 3. Describe sources of error. 4. Suggest further research. P a g e | 15 Identifying the organic compounds in different food sources Pre-Lab Discussion The most common organic compounds found in the living body are lipids, carbohydrates, proteins, and nucleic acids. Common foods, which often consist of plant materials or substances derived from animals, are also combinations of these organic compounds. Simple chemical tests with substances called indicators can be conducted to determine the presence of organic compounds. A color change of an indicator is usually a positive test for the presence of an organic compound. Objective Use several indicators to test for the presence of lipids, carbohydrates, and proteins in particular foods Materials 10 Test tubes Test Tube rack Masking tape Hot plate Iodine solution 20 ml honey solution 20 ml egg white solution 20 ml corn oil 20 ml lettuce solution 20 ml gelatin solution 20 ml potato solution 20 ml melted butter 20 ml distilled water 20 ml apple juice solution Brown paper towels 20 ml unknown substance Biuret Reagent Sudan IV Stain Benedict’s Solution 600 ml beaker Procedure I. TESTING FOR LIPIDS (Paper towel Test, Sudan Stain test) A. Paper Towel Test 1. Obtain 10 test tubes and place them in a test tube rack. Wrap masking tape 1-1/2 times around the tube and label each. Be sure that the tape is fastened properly. Label the paper towel as follows: Honey Egg White Corn Oil Lettuce Gelatin Butter Potato Apple Juice Distilled H2O Unknown 2. Fill each test tube with a “finger width” of the substance indicated. 3. Use your thumb to invert the tube and place a thumbprint of the food on the paper towel in the correct box. The “smear” should be about the size of a quarter. Do not invert the tubes over the paper towel to avoid spillage. Set the paper aside until all other data for the lab is collected. P a g e | 16 4. Come back to this section after all other data has been collected for the lab. Hold the paper towel up to the light. You will notice that some foods leave a translucent spot on the brown paper. The translucent spot indicates the presence of lipids. 5. Place a check mark on the data table for those substances, which have shown the presence of lipids for the paper towel test. B. Sudan Test for Lipids 1. Add a toothpick scooping of Sudan IV Stain to each of the 10 tests tubes and shake vigorously. CAUTION: Sudan IV will stain skin and clothing. Use caution when handling. Sudan IV stain will change the solution of a reddish color in the presence of lipids. 2. Record the resultant colors and place a check in the appropriate box to indicate the substances, which contain lipids. 3. Wash all test tubes thoroughly. TESTING FOR CARBOHYDRATES (Iodine Test, Benedicts Test) A. Iodine Test 1. Refill each cleaned test tube with a “finger width” of the substance indicated on the masking tape label. Add 5 drops of iodine solution to each tube and shake. Iodine will change to a dark blackish-blue color in the presence of starch. CAUTION: Iodine will stain skin and clothing. Use caution. 2. In the data table, record the color of all tubes. Place a checkmark beside each tube that contains carbohydrates. 3. Wash all tubes thoroughly. B. Benedict’s Test (hot water bath) 1. Set up a 600 ml beaker (200 ml of tap water) water bath on a hot plate as shown below. Heat the water bath to a gentle boil. CAUTION: Use extreme care when working with hot water. Do not let the water splash onto your hands. 2. While the water bath is heating, fill each of the clean tubes with a “finger width” of the substance on the masking tape. Then add 10 drops of Benedict’s solution to each of the tubes. 3. Place the tubes in the heated water bath for 2 minutes. 4. Using test tube holders, remove the tubes from the bath. When heated, Benedict’s solution will change color to a muddy, rusty orange-ish color in the presence of carbohydrates. Record all test tube colors in the data table and place a check by those foods that contain carbohydrates. 5. After they have cooled, wash the test tubes thoroughly. TESTING FOR PROTEINS (Biuret’s Test) A. Biuret’s Test 1. Place a “finger width” of the appropriate substance in each of the labeled test tubes. Add 10 drops of Biuret reagent to each tube. CAUTION: Biuret contains a strong base. If you get it on your skin, wash it off immediately with water. Shake the tubes vigorously. P a g e | 17 2. Biuret reagent changes to a blue-violet color in the presence of protein. In the data table, record all test tube colors and place a check mark next to any substances that contain proteins. 3. Wash the test tubes thoroughly. (Finish your paper towel lipid test.) Observations Data Substance Lipid Tests Carbohydrate Tests Protein Test Complex Simple Paper Lipids Iodine Biuret Proteins Sudan Color Carbs Benedicts Color Carbs Towel Present? Color Color Present? present? Present? Honey Egg White Corn Oil Lettuce Gelatin Butter Potato Apple Juice Distilled Water Unknown Discussion 1. Which test substances contain lipids? 2. Which test substances contain carbohydrates? 3. Which test substances contain protein? 4. Which test substance did not test positive for any of the compounds? 5. What is the purpose of using distilled water as one of your test substances? 6. What do all indicators have in common? What do indicators do? P a g e | 18 7. Your brown bag lunch has a large, translucent spot on the bottom. What explanation could you give for this occurrence? 8. What conclusion could you make if a positive test for any of the organic compounds occurred in the test tube containing only distilled water? 9. A very thin slice is removed from a peanut and treated with Sudan stain. Then a drop of Biuret reagent is added to the peanut slice. When you examine the peanut slice under the microscope, patches of red and blue violet are visible. What conclusions can you draw from your examination? 10. When a police officer determines that a homicide had occurred on some individual, then they may request that the food found in their victim’s stomach be tested. We in fact tested this food as our unknown. So where should we tell the police that the victim had eaten. Did the victim just eat at a greasy burger at the City Diner, fettucini alfredo at the Italian pasta house, or barbeque chicken from Hickory Farm? Explain your reasoning. 11. Why would someone on a diet eat lettuce? Explain. P a g e | 19 What are the effects of different wavelengths of light on photosynthetic rate? Objective Determine how different colors of light effect photosynthetic rate Materials list (per group) 1-Ringstand 1-O-Ring 1-light 1-shoebox (provided by students) 1 -4” x 4”squares of colored cellophane (red, blue, and green) 5-150 ml beakers 160 ml 0.2% Sodium bicarbonate solution (40 ml per beaker) 50 spinach disks (spinach provided by instructor) with O2 removed Procedure – Part I: Preparing the spinach disks 1. Each group of 4 students needs to prepare 50 spinach disks total for their 5 treatments. Stack the spinach leaves on the desk and punch holes in them using the potato core device. Be sure to punch about 10 extra for the experiment. Discard any incomplete “holes”. 2. Place all spinach disks into a sodium bicarbonate vacuum for 20 minutes. This removes the Oxygen from the disks. Once removed from the vacuum all of the disks should sink to the bottom of the Erlenmeyer flask. Repeat for additional time if necessary. Procedure – Part II: Setting up the experiment 1. Cut three 3” x 3” squares in the bottom of the shoebox as instructed by your teacher. Cut 3 different colors of cellophane (red, blue, green) and tape them over the openings in the box. The fourth setup will receive white light and the fifth will not receive any light. 2. Next, divide the inside of the shoebox into 6 even sections using construction paper. 3. Fill each beaker with 40 ml of sodium bicarbonate solution. Place 10 oxygen removed spinach disks in each beaker. (They should sink to the bottom. If they do not, then discard, and get additional disks). 4. Invert the shoebox and cover the beakers. Each beaker should be located in a different quadrant of the shoebox. P a g e | 20 5. Set up the ring stand, o-ring, and light as instructed by your teacher. At what distance should the light be placed? (Caution: Do not put the light too close to the shoebox or it may catch fire.) 6. Wait 50 minutes and then remove the shoebox. Without disturbing the beakers, count the number of disks that are floating (i.e., not 100% flat on the bottom of the beaker). 7. Create a class data table and record the percent of floating disks in found in each color of light for the entire class. 8. Clean up the work area and analyze the class data. Discussion 1. What is the purpose of using the beaker of disks in the dark? 2. What color of light produced the highest percent of floating disks? 3. Which wavelength of light was most effective in promoting photosynthesis? Why? 4. Which wavelength of light was least effective in promoting photosynthesis? Why? 5. Was your initial hypothesis supported or rejected? Why? 6. Describe at least 2 sources of error and 2 ways to improve this experiment. P a g e | 21 Respiration of Sugars by Yeast Introduction 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. It must also have the proper enzymes capable of breaking the food’s chemical bonds in a useful way. Sugars are vital to all living organisms. Yeast 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. In this lab, you will try to determine whether yeast are capable of metabolizing a variety of sugars. When yeast respire aerobically, oxygen gas is consumed and carbon dioxide, CO2, is produced. You will use a CO2 Gas Sensor to monitor the production of carbon dioxide as yeast respire using different sugars. The four sugars that will be tested are glucose (blood sugar), sucrose (table sugar), fructose (fruit sugar), and lactose (milk sugar). Objectives Use a CO2 Gas Sensor to measure concentrations of carbon dioxide Determine the rate of respiration by yeast while using different sugars Determine which sugars can be used as a food source by yeast CO2 Gas Sensor Figure 1 Materials computer 600 mL beaker (for water bath) Vernier computer interface Beral pipettes Logger Pro hot and cold water Vernier CO2 Gas Sensor thermometer 250 mL respiration chamber four 10 100 mm test tube 5% starch, glucose, sucrose, lactose, and yeast suspension fructose sugar solutions P a g e | 22 Procedure 1. Obtain six test tubes and label them T, G, S, F, L, and W. 2. Obtain the five sugar solutions: starch, glucose, sucrose, fructose, and lactose. a. Place 2 mL of the starch solution in test tube T. b. Place 2 mL of the glucose solution in test tube G. c. Place 2 mL of the sucrose solution in test tube S. d. Place 2 mL of the fructose solution in test tube F. e. Place 2 mL of the lactose solution in test tube L. f. Place 2 mL of distilled water in test tube W. 3. Obtain the yeast suspension from the water bath. The water bath ensures that the yeast will remain at a constant and controlled temperature (38°C). Gently swirl the yeast suspension to mix the yeast that settles to the bottom. Put 2 mL of yeast into each of the six test tubes. Gently swirl each test tube to mix the yeast into the solution. 4. Set the six test tubes into the water bath. 5. Incubate the test tubes for 10 minutes in the water bath. Keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove as much water as you will add, or the beaker may overflow. Use a beral pipet to remove excess water. While the test tubes are incubating, proceed to Step 7. 6. Connect the CO2 Gas Sensor to the computer interface. Prepare the computer for data collection by opening the file “12A Yeast Respiration” from the Biology with Vernier folder of Logger Pro. 7. When incubation is finished, use a beral pipet to place 1 mL of the solution in test tube T into the 250 mL respiration chamber. Note the temperature of the water bath and record as the actual temperature in Table 1. 8. Quickly place the shaft of the CO2 Gas Sensor in the opening of the respiration chamber. Gently twist the stopper on the shaft of the CO2 Gas Sensor into the chamber opening. Do not twist the shaft of the CO2 Gas Sensor or you may damage it. 9. Begin measuring carbon dioxide concentration by clicking . Data will be collected for 4 minutes. 10. When data collection has finished, remove the CO2 Gas Sensor from the respiration chamber. Fill the respiration chamber with water and then empty it. Make sure that all yeast have been removed. Thoroughly dry the inside of the chamber with a paper towel. P a g e | 23 11. Determine the rate of respiration: a. Move the mouse pointer to the point where the data values begin to increase. Hold down the left mouse button. Drag the pointer to the end of the data and release the mouse button. b. Click on the Linear Fit button, ,to perform a linear regression. A floating box will appear with the formula for a best fit line. c. Record the slope of the line, m, as the rate of respiration in Table 1. d. Close the linear regression floating box. e. Share your data with the class by recording the sugar type and respiration rate on the board. 12. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu. 13. Use a notebook or notepad to fan air across the openings in the probe shaft of the CO2 Gas Sensor for 1 minute. 14. Repeat Steps 8 – 14 for the other five test tubes. Data Table 1: Individual Results Sugar Tested Respiration Rate (ppm/min) Starch Glucose Sucrose Fructose Lactose Water (control) Table 2: Class Averages Sugar Tested Respiration Rate (ppm/min) Starch Glucose Sucrose Fructose Lactose Water P a g e | 24 Results 1. When all other groups have posted their results on the board, calculate the average rate of respiration for each solution tested. Record the average rate values in Table 2. 2. On Page 2 of the experiment file, make a bar graph of rate of respiration vs. sugar type. The rate values should be plotted on the y-axis, and the sugar type on the x-axis. Use the rate values from Table 2. Discussion 1. Considering the results of this experiment, do yeast equally utilize all sugars? Explain. 2. Why do you need to incubate the yeast before you start collecting data? 3. Why could certain carbohydrates be used successfully for respiration by yeast, and others not? 4. How does the absence or presence of certain enzymes in yeast can affect their ability to use certain carbohydrates? 5. How are monosaccharides, disaccharides and polysaccharides used differently by yeast cells? 6. How is sugar processed differently in yeast in the absence of oxygen. 7. Why would all yeast would be lactose intolerant, while only some humans are considered lactose intolerant. 8. Yeast lives in many different environments. Make a list of some locations where yeast might naturally grow. Estimate the possible food sources at each of these locations P a g e | 25 Making Root Beer A Practical Example of Anaerobic Respiration Introduction History of Root Beer: Root beer was made by our forefathers by soaking Sasafras (a type of tree) root in water, and adding sugar and yeast (for carbonation). In the early 1900's however, scientists discovered that safrole, a chemical found in Sassafras root, was a carcinogen (which means it is a cancer causing agent.) Now, a mixture of other herbs and spices makes up "root beer extract" which is what we now use to make homemade root beer. Background Information: There are two types of respiration: aerobic (requiring oxygen) and anaerobic (without oxygen). Yeast cells (a type of fungus) obtain energy from glucose (sugar) by a specific anaerobic process called fermentation. There are two types of fermentation, lactic acid fermentation (which occurs in muscle cells when they are oxygen deprived), and alcoholic fermentation, which is involved in the making of food products. Alcoholic fermentation begins after glucose diffuses into the yeast cell. The glucose is broken down into two 3-carbon molecules called pyruvic acid. The pyruvic acid is then converted to CO2, ethanol, and energy for the yeast cell. Don't get excited, students, there is very little ethanol in this root beer. Doesn’t anaerobic fermentation produce ethanol? The answer is yes, but... The alcoholic content which results from the fermentation of this root beer and found it to be between 0.35 and 0.5%. Comparing this to the 6% in many beers, it would require a person to drink 1-5 gallons of this root beer to be equivalent to one 12 ounce alcoholic drink. I would call this amount of alcohol negligible, but for persons with metabolic problems who cannot metabolize alcohol properly, or religious prohibition against any alcohol, consumption should be limited or avoided. However, there are many high school biology labs that have made this beverage without any problems. Fermentation is used to make a variety of food products, including the making of wine, bread, cheese, sauerkraut, and baked goods. It is the carbon dioxide produced by the yeasts that give root beer its "fizz." This fizz is produced in store bought root beer by a carbonation machine that forces carbon dioxide into the root beer mixture, without the aid of our little yeast friends. Materials (All materials should be clean materials used for food preparation) 2-Liter, clean plastic bottles Funnel Bottled spring water (or tap water left out for 24 hours) Dry baker’s yeast Root beer extract Sugar Electronic Balance Weighing Boats/Paper Plastic Spoons 25-mL Graduated Cylinder P a g e | 26 Procedure 1. Gather the materials. 2. With a dry funnel, add in sequence: 200 g of table sugar (You can adjust the amount to achieve the desired sweetness (no more than 300 g.) 3. Add: 0.8 g powdered baker's yeast. You can see the yeast granules on top of the sugar. Do not alter the amount of yeast. 4. Shake to distribute the yeast grains into the sugar. 6. Add any other ingredients you feel necessary to create the ultimate root beer. 7. Add with a dry funnel: 15 mL of root beer extract (Zatarain's or Hires, etc) 8. The extract sticks to the sugar which will help dissolve the extract in the next steps. 9. Half fill the bottle with bottled spring water. If you are using tap water, allow the water to sit for 24 hours to allow the chlorine to dissipate. Rinse in the extract which sticks to the tablespoon and funnel. Swirl to dissolve the ingredients on top of the dry sugar. 10. Fill to the neck of the bottle with fresh cool tap water, leaving about an inch of head space, securely screw cap down to seal. Invert repeatedly to thoroughly dissolve the contents. 11. Place at room temperature for 24 hours or until the bottle feels hard to a forceful squeeze. NOTE: Do not leave the finished root beer in a warm place once the bottle feels hard. After a couple weeks or so at room temperature, especially in the summer when the temperature is high, enough pressure may build up to explode the bottle! There is no danger of this if the finished root beer is refrigerated. 12. Move to a cool place (below 65°F). Refrigerate overnight to thoroughly chill before serving. Crack the lid of the thoroughly chilled root beer just a little to release the pressure slowly. NOTE: There will be a sediment of yeast at the bottom of the bottle, so that the last bit of root beer will be turbid. Decant carefully if you wish to avoid this sediment. Discussion 1. After 24 hours, observe your root beer bottle. What has happened? Why? 2. What is alcoholic fermentation? What happens during this process? 3. What factors do you think might affect the rate of fermentation? (Name at least three. 4. How did your root beer taste compared to other groups? Would you do anything differently if you were to make homemade root beer again? Explain. P a g e | 27 Case Studies - Trophic Levels Introduction One scenario will be introduced (at a time) to the class by the teacher. Each student group (consisting of 4 students) will present their consensus on the scenario, explaining the science and bias behind their group decision. Groups are meant to have different solutions and viewpoints. Each group of 4 students will represent one of the viewpoints listed below. The groupings are as follows: Farmers/gardening enthusiasts Parents of kids and pet owners Police officers/Firemen/EMT Animal control personnel Scientists/Environmentalists Senior citizens Scenario #1: In Mt. Lebanon, many citizens are concerned about the overabundant deer population. The deer are eating gardens and shrubbery. The deer numbers have been increasing over the past decade. Many are unhealthy and there are increased automobile accidents. In order to keep them under control, someone proposed introducing predators, such as tigers, to Mt. Lebanon. However, city council needs voter support to secure funding for this. What are some of the concerns and potential solutions to the deer problem? Scenario #2: Some people dream of genetically reincarnating extinct species (Dinosaurs, Wooly Mammoths, Asian Oxen, Gaurs, and Saber Tooth Tigers). Ideas like sperm freezing or digging up preserved animals that still contain DNA may make this a reality. Some suggest using currently living animals as the surrogate mothers. What are the benefits and concerns about bringing back extinct species? Scenario #3: Many farmers and gardening enthusiasts have their vegetation threatened by spider mites, aphids, leaf miners, and beetles. To stop these insects from eating the leaves off of their plants, they want to introduce lady bugs to eat these insects instead of spraying pesticides. Is the introduction of a predator a better option than pesticides? What are the benefits and concerns? Scenario #4: Some groups want to use 50% of current corn crops for creation of biofuels (to be used in powering vehicles and for heating). Do you think that biofuels are a viable energy source? What are the benefits and concerns? P a g e | 28 Scenario #5: If the climate of an area changes (such as due to global warming), how will that affect the energy and matter flow of the ecosystem? For example, polar bear habitats are shrinking, putting them at risk of extinction. Savannahs and grasslands are becoming deserts. What are some of the concerns and potential solutions to climate change and its effects? Scenario #6: Pymatuning Lake is a popular lake north of Pittsburgh for tourists. Among other outdoor activities, tourists can feed the carp at the spillway between Pymatuning Lake and Sanctuary Lake. The state is banning the feeding of bread to the overpopulated carp in order to reduce the amount of nutrient input into the lake, such as phosphates and nitrates, which have resulted in increased aquatic plant growth. Visitors will have to purchase fish food, primarily made of ground corn, in place of using bread. What are some of the concerns with the proposal? What alternative solutions are there? Scenario #7: Pennsylvania Game Commission issues a specified number of hunting permits for various animals each year. Why do they pick certain species? Why do they limit the number of permits? For example, it is quite rare to see a black bear in the South Hills. However, citizens are still issued permits for hunting bear. Why? Also, which animals in food webs/ chains should be permitted to be hunted (if any)? P a g e | 29 How Do Cell Membranes Regulate Transport? Part I. How do concentrations of solute inside a cell affect the rate and direction of osmosis? Objective Determine how the concentration of solute within a cell affects the rate and direction of osmosis Materials 5 - 250ml beakers 5 dialysis tubes String Electronic Balances Distilled Water 20% Sucrose Solution 40% Sucrose Solution 60% Sucrose Solution Procedure 1. Obtain 5 beakers to represent the cellular environments and 5 dialysis tubes to represent cells. 2. Set up the beakers and dialysis tubes as according to the table below: Beaker Dialysis Tube Distilled Water Distilled Water Distilled Water 20% Sucrose Distilled Water 40 % Sucrose Distilled Water 60% Sucrose 20% Sucrose Distilled Water **Sucrose cannot diffuse through the dialysis tube 3. To create your model cells: a. Obtain a piece of pre-soaked dialysis tubing. Tie one tight knot in one end of the tubing. Leave the other end open. b. Add the appropriate substances into the tubing. Make sure the bag is moist. You may need to rub the untied end between your fingers to open it. c. Close, gently press out air bubbles, and knot the open end of each bag. Make sure to leave room in your bag for growth. Remember, “cells” may gain or lose mass! Do not mix up your bags. You may wish to use a numbered paper towel for each setup. 4. Measure and record the initial mass of each bag. 5. After placing the cells in their environments, run your experiment for 30 minutes and record your final mass. 6. Determine the percent mass change in a half hour for each “cell”. a. % mass change = (final mass - initial mass)/initial mass x 100 7. You may set up Part II concurrently with Part I. Do not totally abandon Part I if you are taking regular measurements. P a g e | 30 Part II. How large are the pores of a selectively permeable membrane? Objectives Determine the size of the pores of a selectively permeable membrane Observe diffusion of different substances using indicators Materials 1 - 250ml beaker 1 dialysis tube String Electronic Balances Distilled Water Glucose Tes-Tape (Glucose) Starch Solution Iodine Procedure 1. Obtain 1 beaker to represent the cellular environment and 1 dialysis tube to represent a cell. 2. You have available 3 different sized molecules – Glucose, Iodine, and Starch. You also have access to a glucose test. 3. Design an experimental setup, using the indicators (iodine and glucose test paper) to show if the different molecules can pass through the membrane. Have you teacher approve your design. 4. Create your cell model using the procedure from Part I. 5. After placing the cell in its environment, run your experiment for at least 10 minutes. 6. Use the data table to record your initial and final observations. Data Distilled Water Distilled Water Distilled Water Distilled Water 20% Sucrose Part I Beaker/Distilled Beaker/20% Beaker/40% Beaker/60% Beaker/Distilled Water Cell Sucrose Cell Sucrose Cell Sucrose Cell Water Cell Initial mass Mass after 30 min. Percent Mass Change Total Mass Change = Final Mass - Initial Mass Percent Mass Change = (Total Mass Change/Initial Mass) x 100 Part II Initial Observation Final Observation Did the molecules move through the Cell Beaker Cell Beaker membrane? Iodine Glucose Starch P a g e | 31 Results and Discussion Use the following questions to help you summarize each experiment. Part I: Was there correlation between concentration and rate of osmosis? Was there correlation between concentration and direction of osmosis? How do you know? Create a graph to show your data. Part II: Explain your reasoning for the movement or lack of movement of each of the molecules. What evidence do you have? Are the “cells” selectively permeable? Can you determine the size of the pores in the bags? Application 1. Look at the picture below to answer questions a, b, c, and d. a. Which substance(s) will rapidly enter the cell? b. Which will rapidly leave the cell? c. Which will neither enter nor leave the cell? Explain. 2. Sucrose cannot pass through the membrane. Based on what you learned in Part I, what would you expect to happen to the “cell’s” mass if: a. you place a “cell” of 40% sucrose into a cup with 20% sucrose? b. you place a “cell” of 40% sucrose into a cup with 40% sucrose? c. you place a “cell” of 40% sucrose into a cup with 60% sucrose? 3. An animal cell is placed in an environment that contains a higher concentration of water than the cell itself. This type of environment is called a hypotonic environment. What would happen to the animal cell in terms of water movement? 4. A plant cell is placed in an environment that contains a lesser concentration of water than the cell itself. This type of environment is called a hypertonic environment. What would happen to the plant cell in terms of water movement? P a g e | 32 What environmental factors affect xylem transport in plants? Introduction One of the reasons that even wet summers can still end in drought is the efficiency of plants at moving water from the ground to their upper reaches where it evaporates from the surface of the foliage. The process is called transpiration and it's essential for moving water to all parts of plants, even to the tops of the tallest trees. Celery, a vascular plant, has special tubes, called xylem, to take up water. You can imagine with the tallest trees that each water-carrying xylem contains a continuous, thin column of water over a hundred feet in length and reaching from the roots to the uppermost leaves. The effect of evaporation at the top of the tree literally pulls this column of water up the tree. The ability of these thin columns of water to be pulled in this way – without breaking – is attributed to the cohesive forces between the water molecules in the liquid; this is called capillary action. Weather is a stimulus that affects plant transpiration. For example, the process of transpiration is accelerated by evaporation from the celery leaves. Other environmental factors also affect the rate of transpiration. You will investigate these factors. Objective Determine how different environmental factors affect xylem transport in celery. Materials Tub of water Red and Blue Food Coloring Each group of students will need the following: Gooseneck lamp Four 125mL flasks Four Stalks of celery Metric ruler China marker Razor blade Optional materials: Salt Sugar Vinegar Corn Syrup Table fan Hair Dryer Hot plate Ice Cubes Procedure 1. Determine three variables you wish to test. Remember to also set up a control as your fourth setup. You must obtain the basic set of materials for each group, as well as any other materials you will need. Consider the following variables: Chemical composition, viscosity, pH, light, wind, temperature, and foliage 2. Using a razor blade, cut four stalks of celery off while they are submerged underwater. It is important to do this underwater to ensure that no air enters the xylem. P a g e | 33 3. Immediately transfer the stalks to the four flasks containing the appropriate dye and solution (if necessary). Make sure each flask is labeled for each variable. Determine the length of time to run the experiment and begin timing. Results Create a data table to record any information that you may collect. You must also present your data graphically. Discussion 1. Explain how each setup is representative of an environment or scenario. Explain what materials you used to simulate that environment. 2. How does transport in each compare to the control? 3. How do different environmental factors affect xylem transport in celery? Be sure to explain your reasoning. 4. Give real life examples of how plants are able to survive in environments similar to what you tested. What adaptations do plants have? P a g e | 34 What are the effects of exercise on CO2 output and heart rate? Objectives Determine the effect of exercise on CO2 output and heart rate Determine the effect of different exercises on CO2 output and heart rate Materials (per group) 400-600 mL 0.004% Bromthymol blue solution 2 – 500 mL Ehrlenmyer flasks 1 Solid #7 Rubber Stopper Glass stirring rod Drinking straw 100 mL Graduated cylinder 4% ammonia dropper bottle Clock or stop watch with second hand Vernier Equipment with Heart Rate Monitor Procedures After each part of this Investigation, rest and allow your heart rate and breathing rate to return to normal. CAUTlON: If at any time during this investigation you feel faint or dizzy sit down and immediately call your teacher. Experimental Requirements You must test at least two experimental treatments You must have a control You must have a minimum of three replicates per treatment Follow procedures A and B when appropriate A. Procedure to measuring CO2 output using bromthymol blue 1. Pour 100 mL of bromthymol blue solution into your flask. 2. Have one member of your group use a drinking straw to breathe out normally into the bromthymol blue solution for exactly one minute. CAUTION: Be careful not to suck the solution into your mouth. The bromthymol blue solution should turn a pale yellow color at the end of one minute. 3. With a medicine dropper, add 1 drop of ammonia to the flask and stir once with a glass stirring rod. 4. Continue to add ammonia one drop at a time. Count each drop and stir once between drops until the solution turns blue. In a data table, record the total number of drops of ammonia needed to turn the solution blue. 5. Replenish your solution for each trial. P a g e | 35 B. Procedure for using a hand-grip heart rate monitor 1. Grasp the handles of the Hand-Grip Heart Rate Monitor. Place the fingertips of each hand on the reference areas of the handles (see Figure 1). 2. The left hand grip and the receiver are both marked with an alignment arrow. When collecting data, be sure that the arrow labels on each of these devices are in alignment (see Figure 2) and that they are not too far apart. The reception range of the plug-in receiver is 80–100 cm, or ~3 feet. Figure 1 Figure 2 3. Prepare the computer for data collection by opening the file “27 Heart Rate & Fitness” from the Biology with Vernier folder of Logger Pro. 4. Click to begin monitoring heart rate. 5. Click when you have determined that the equipment is operating properly. Results Construct appropriate data tables Graphical representation Discussion 1. What does the color change of bromthymol blue represent? 2. Is there an effect of exercise on CO2 output and heart rate? If so, what is the effect and why does it happen? 3. Is there relationship between CO2 output and heart rate? If so, what is the relationship and why is there one? 4. What are the effects of different forms of exercise on CO2 output and heart rate? P a g e | 36 What Can You Learn By Testing Urine? Introduction All organisms produce wastes. These waste materials must be removed so that the organism is not poisoned by its own metabolism. In humans, urine is the fluid produced by the kidneys as they remove waste chemicals from the blood. Urine is made up primarily of water, with some salts and organic materials dissolved in it. The concentration of each of these substances varies with a person’s health, diet, and degree of activity. By testing the chemical composition of urine, doctors can learn much about the general health of an individual. Urinary tract infections, kidney malfunction, drug abuse, diabetes, and liver disease are just some of the medical problems that can be diagnosed through urinalysis. Urinalysis involves the physical, chemical, and visual examination of a urine sample. Objectives Perform several tests to detect substances in a sample of artificial urine Determine the contents of an artificial sample of unknown composition Problem What can urine testing tell you about a person’s health? Materials (per group) Urine sample without glucose Urine sample with glucose Urine sample without chloride Urine sample with chloride Urine sample without phosphate Urine sample with phosphate Urine sample without albumin Urine sample with albumin 0.1 M Silver nitrate (AgNO3) 0.1 M Barium chloride (BaCl2) 5 test tubes Glass-marking pencil Test tube rack Test tube holder 10 mL graduated cylinder Hot plate 400 mL beaker Benedict’s solution Biuret’s solution Safety Put on a laboratory apron and safety goggles. Handle glassware carefully. Use extreme care when working with heated equipment or materials to avoid burns. Laboratory chemicals may irritate the skin or cause staining of the skin or clothing. Never touch or taste any chemical unless instructed to do so. Discard all solutions as indicated by your teacher. P a g e | 37 Procedures Part A: Testing for Glucose 1. Use a 600 mL beaker and hot plate to prepare a hot water bath. 2. Place two test tubes in a test tube rack. Label one as “experimental” and one as the “control.” 3. Add 5 ml of urine with glucose to the correct tube. Add 5 ml of control urine to the other tube. 4. Add 5 drops of Benedict’s solution to both test tubes. Record the pre-heated color of the test tubes in the data section. 5. Place both test tubes in the hot water bath for 3 minutes then remove the test tubes with a test tube holder. Place the test tubes in the test tube rack. 6. Record any color changes in the test tubes in the data section. 7. Dispose of the solutions in the sink with plenty of water. Wash the tubes thoroughly. Part B: Testing for Albumin 1. Place two test tubes in a test tube rack. Label one as “experimental” and one as the “control.” 2. Add 5 ml of urine with albumin to the correct tube. Add 5 ml of control urine to the other tube. 3. Add 5 drops of Buiret’s solution to both test tubes. 4. Record any color changes in the test tubes in the data section. 5. Dispose of the solutions in the sink with plenty of water. Wash the tubes thoroughly. Part C. Testing for Chloride (2-tests) Silver Nitrate Test 1. Place 2 test tubes in a test tube rack. Label one as “experimental” and one as “control.” 2. Add 5 ml of urine with chloride to the correct tube and 5 ml of control urine to the other tube. 3. Add 3 drops of Silver Nitrate (AgNO3) to both tubes. Observe the top surface and overall color of the liquid in each test tube. Record your observations in the data section of the lab. 4. Dispose solutions in waste beaker labeled “Silver Waste”. Do not pour these solutions down the sink. Wash the tubes thoroughly. Barium Chloride Test 1. Place 2 test tubes in a test tube rack. Label one as “experimental” and one as “control.” 2. Add 5 ml of urine with chloride to the correct tube and 5 ml of control urine to the other tube. 3. Add 3 drops of Barium Chloride (BaCl2) to both tubes. Observe the top surface and overall color of the liquid in each test tube. Record your observations in the data section of the lab. 4. Dispose solutions in waste beaker labeled “Barium Waste”. Do not pour these solutions down the sink. Wash the tubes thoroughly. P a g e | 38 Part D. Testing for Phosphate (2-tests) Silver Nitrate Test 1. Repeat the methods for the silver nitrate test from Part C. Barium Chloride Test 1. Repeat the methods for the barium chloride test from Part C. Part E. Testing an Unknown Urine Sample 1. Put 4 test tubes in a test tube rack. Label one test tube for the glucose test, one for the albumin test, and 2 for the chloride/ phosphate test. 2. Fill each test tube with 5 ml of unknown urine. 3. Perform each of the tests in section A-C in the lab to determine which substances are in the unknown urine. The test for part D is the same as part C so you do not need to repeat it. 4. Record your findings in the data section of the lab. 5. Dispose of the solutions accordingly and remove the tape from the test tubes. 6. Thoroughly wash the test tubes and return them to their original location. Results Data Table 1: Glucose Test Substance Color Before Heating Color After Heating Glucose Urine Control Data Table 2: Chloride Test Substance Color before Color after Color before Color after adding adding adding BaCl2 adding AgNO3 AgNO3 BaCl2 Chloride Urine Control Chloride Urine Control P a g e | 39 Data Table 3: Phosphate Test Substance Color before Color after Color before Color after adding adding adding BaCl2 adding BaCl2 AgNO3 AgNO3 Phosphate Urine Control Phosphate Urine Control Data Table 4: Albumin Test Substance Color Before Biuret’s Color After Biuret’s Solution Solution Albumin Urine Control Data Table 5: Unknown Urine Sample Test Color After Test Present or Absent? Glucose Albumin Chloride Phosphate Discussion 1. Which substance(s) did you find in your unknown urine sample? Do you think this sample came from a healthy person or sick person? Explain your answer. 2. a) What is diabetes? Explain the difference between hypoglycemic and hyperglycemic. P a g e | 40 b) If you add Benedict’s solution to a urine sample from a person who has diabetes and heat it, what color would you expect the heated sample to be? Explain your answer by relating it back to your data table and the previous question and base your response on what chemical(s) you expect to be in the urine. 3. While a blood sample contains glucose, phosphate, albumin, and chloride molecules, a normal urine sample contains only phosphate and chloride molecules. Why aren’t glucose and albumin normally found in urine? 4. If a doctor finds high levels of protein in a patient’s urine sample, the doctor will probably test the patient’s urine several times over a week before drawing any conclusions. What might be the reason for this? 5. Why can some chemicals be detected in your urine a month after taking them into your body? 6. Explain sources of error that may have led you to get inaccurate results. 7. What can urine testing tell you about a person’s health? Provide at least three examples to support your claim. P a g e | 41 Which antibiotic or disinfectant is best at controlling E. coli growth? Objective Determine the effects of antibiotics and disinfectants on E. coli growth Materials (per group) Sharpie Bunsen Burner Flint Striker or matches Sterile cotton swab Sterile paper disks Forceps Metric ruler Scotch tape E. Coli culture Sterile nutrient agar plate Various antibiotics Various disinfectants Procedures Part A: Inoculating the Sterile Nutrient Agar Plate 1. Label the 4 quadrants on the bottom of the sterile nutrient agar plate using a Sharpie. See the associated illustration. Note: place the numbers near the edges of the dish so that we can easily see what is happening in each quadrant. 2. Use a sterile swab and obtain a culture of E. Coli. Note: Do not put the cap of the container on the desk when obtaining the culture. 3. Slightly open the sterile agar plate and streak the plate in one direction as you move the swab back and forth. Then, turn the plate 90o and streak the plate in the other direction. See the illustration to the right. 4. Hold the tip of the cotton swab over a flame until it catches fire and then rinse it with water. Place the swab in the appropriate disposal container. Part B: Controlling the Spread of E. Coli with Disinfectants and Antibiotics 1. Examine your agar plate. You should see streaks of bacteria covering the surface of the agar. 2. Light the Bunsen burner and sterilize the forceps. 3. Select 2 disinfectants, 1 antibiotic, and distilled water as the substances that you will test. 4. Obtain a sterile disk and dip it into the container containing one of the disinfectants or distilled water. Note: The antibiotics already come on sterile disks. You do not need to get a sterile disk when gathering your antibiotic. 5. Shake the excess liquid off of the disk. Slightly open the sterile agar plate and place the disk in the center of the appropriate quadrant. Give it a gentle press so that it stays in place. Try not to crack the agar. 6. Repeat steps 2-4 when you place the other disks on the plate. 7. Seal the entire perimeter of the plate with tape. Write your name(s) on the side of the dish as well. 8. Place the nutrient agar dishes in an incubator set at 37 oC overnight and examine the results tomorrow. 9. Be sure to wash your lab area with a disinfectant. Wash your hands thoroughly with soap and water. Part C: Measuring the Zone of Inhibition 1. Remove the plates from the incubator and measure the zone of inhibition for each of the 4 disks placed in the bacteria. If no zone is apparent, record the measurement as zero. 2. Collect the data from the class and determine the average zones of inhibition for each of the products used. 3. Dispose of the Petri dish in the appropriate trash container. 4. Be sure to wash your lab area with a disinfectant. Wash your hands thoroughly with soap and water. P a g e | 42 Results ZONES OF INHIBITION: Effects of Disinfectants and Antibiotics on E. coli Group 1 Group 2 Group 3 Group 4 AVERAGE Disinfectant (mm) (mm) (mm) (mm) (mm) 1. 2. 3. 4. 5. 6. 7. 8. Group 1 Group 2 Group 3 Group 4 AVERAGE Antibiotic (mm) (mm) (mm) (mm) (mm) 1. 2. 3. 4. Group 1 Group 2 Group 3 Group 4 AVERAGE Distilled Water (mm) (mm) (mm) (mm) (mm) Discussion 1. Why is it important to use sterile techniques while inoculating the agar plates? 2. Which disinfectant or antibiotic was most effective in removing the bacteria? 3. If a disinfectant is the most effective, why aren’t they used to treat human bacterial infections? 4. How can you relate this lab to your own life? Discuss what the results mean for antibiotic use, disinfectant use, and the evolution of bacteria. P a g e | 43 How do germinating seeds respond to environmental changes? Cotyledon Shoots Roots Root Hairs Objective To investigate how germinating seeds respond to environmental changes Materials (per group) 2 Petri dishes Scissors Glass marking pencils Nonabsorbent cotton Heavy blotting paper Transparent tape Modeling clay 8 soaked corn grains (or other large seed such as radish seeds) Procedure This is simply a sample procedure to investigate phototropism. To investigate thigmotropism, the students can apply small pieces of tape to the stems of the germinating plants. For gravitropism, the petri dishes can be inverted. For a control, the teacher can construct a few petri dishes so that later the students can compare their results. 1. One day before the investigation, soak the seeds in water. 2. Place 4 water soaked corn grains in the bottom half of a Petri dish. Place one at the top, one at the bottom, and one on both the left and right side. Arrange them so that the pointed portion of the seed faces the center of the dish. 3. Cut a piece of blotting paper slightly larger than the bottom of the Petri dish, wet it thoroughly, and fit it snugly over the grains. 4. Fill the remaining space with wads of nonabsorbent cotton to a depth slightly greater than the thickness of the grains. Do not use too much cotton. 5. Hold the dish on its edge, and observe the grains through the bottom. If they do not stay in place, then pack them with more cotton. 6. When the grains are secure in the dish, seal the two halves of the Petri dish together with tape. 7. Rotate the dish until one of the grains is at the top. With a glass marking pencil, write an “A” on the Petri dish, then proceeding clockwise, label the other grains B, C, and D. Also label the Petri dish with one of the lab member’s last name. 8. Use modeling clay to support the dish on its edge with grain “A” at the top. Place the dish into dim light. 9. Be sure to wash your hands and workstation, and clean up any extra materials. 10. You must determine how you will collect data. When the grains begin to germinate, include sketches and measurements every day for 5 days, showing the direction in which the root and shoot grow from each grain. P a g e | 44 Sensory Receptors Lab Observations In this lab, you will be guided through a series of tasks that have to do with your senses. You will need the help of your lab partner to perform many of these tasks, so make sure you work together. For each task or station, note your observations and your scientific explanations or hypotheses that may explain these observations. Pupil Reflexes: Observations: (include drawings) Explanations: Visual Accommodation Observations: Explanations: P a g e | 45 Blind Spot Observations: Explanations: Binocular Vision Observations: (include drawings) Explanations: Tube: Fingers: Eye Dominance Observations: Explanations: P a g e | 46 Optical Illusions Observations: Explanations: Cars: Animals: Squares and Lines: Hat: Circles: P a g e | 47 Retaining Images Observations: Explanations: Auditory Acuity Observations: Explanations: Sound Localization Observations: Explanations: P a g e | 48 The Sense of Smell Observations: Explanations: The Sense of Taste Observations: Explanations: The Sensation of the Skin Observations: Explanations: Discussion: Using at least three different examples from your investigation, explain how your different senses work together to allow us to respond to stimuli? Include real life situations where these phenomena are applicable. Examples may include things you do on a day to day basis. P a g e | 49 Testing the Nervous System Objectives The purpose of this lab is for each group to answer a question based on an application of the nervous system. Some possible questions that can be asked: Do different variables affect reflex time? Can you control your heart rate consciously? Does short-term memory differ amongst individuals? Methods 1. You and your group will either choose a question from this list or develop a question on you own. Once you have done so, you will indicate to your teacher the question that you have chosen. If you are developing you own question, you must have it approved. 2. Once you have chosen a question, you must first develop a hypothesis based on the question. This will be a prediction based on your question. 3. Next, you will develop an experimental procedure to test you question. This procedure must be redundant enough that it will provide evidence to support your conclusions, so several trials will be necessary. Also, as you develop your experiment, you must also develop ways to collect and analyze data. You will need several data tables to support your conclusions. *You may need to test individuals who are not in your group, so as to have a full data set. 4. Once you have developed your experiment, please have it approved by your teacher to make sure that you have all of the materials that you need. 5. The running of the experiment will come next and allow you to test you hypothesis. Please make sure that all of the data that you record is done in an orderly fashion as to be read later. 6. Once you have concluded your experiment, the most important part of the lab will be to present you conclusions. You will present whether you accept or reject your hypothesis based on your data. You must discuss your experimentation and support your claim with sufficient data. P a g e | 50 Investigating Invertebrate Behavior Objectives Study basic invertebrate anatomy Safely handle invertebrates Design an experiment to study invertebrate behavior Materials Small paintbrush Spoon Ruler Petri dish Stereoscopic dissecting microscope Scissors Tape Optional materials 50 ml beaker with water Paper towels Plain white paper Plastic Bin Assorted rocks Fragrances Food sources Organisms Pill bugs Crayfish Snails Worms Spiders Various insects Procedure I. General Observations Work with at least two organisms of the species being investigated to answer the following questions. 1. Does your organism have any appendages? If so, what are their functions? 2. Does your organism have a hydrostatic skeleton, an endoskeleton, or an exoskeleton? Explain how you know. 3. What does the organism do when it comes to an edge where there is a drop off? 4. Can the organism climb on steep smooth surfaces like the edges of the Petri dish? Can it climb on rough surfaces like your arm? 5. How does your organism seem to sense its environment? 6. Can you tell the difference in males and females? Explain. P a g e | 51 7. Does your organism exhibit dominance behaviors? Explain. 8. How does your organism respire? 9. What are some stimuli your organism seems to respond to? 10. Describe how your organism moves. 11. How fast does your organism move? Make an X in the center of the copy paper. Place the organism on the center of the X. Let it go and record the time in seconds that it takes to run off the paper. Place another X where it left the paper. Measure the distance between the 2 X’s in centimeters. Calculate the speed of the organism in cm/sec. 12. How does your organism respond when disturbed? 13. Do you have all of the same species? How do you know? II. Microscopic Observations using a Dissecting Stereoscope When you make a sketch of the organism, don't just draw an oval with a few squiggly legs - you are expected to do a scientific illustration similar to the sketch of an earthworm below. Include these items in your sketch and be sure to label them on your drawing. Determine the relative proportions (length, width, height as well as lengths of body parts) Count the number of body segments, antennae, and appendages Locate and label the body parts Hair locations Does it have eyes? How many? Compound or simple? Examine the ventral (underside) surface. P a g e | 52 Make your sketch in the space provided: III. Designing and Reporting an Investigation 1. Brainstorm with your partner about possible behaviors you would like to investigate. 2. Select one problem you would like to investigate. 3. Write a hypothesis that relates to your problem. Here is a good and bad example: Poor: I think Pill bugs will move toward the wet side of a choice chamber. Better: If Pill bugs prefer a moist environment, then when they are randomly placed on both sides of a wet/dry choice chamber and allowed to move about freely for 10 minutes, most will be found on the wet side. 4. Identify the variable you will manipulate (independent variable), the variables that will be controlled or stay the same, and one variable will be the one that you will measure (dependent variable). P a g e | 53 Methods For the experiments you design, you will need to create behavior to test the organisms’ reactions. Each basic chamber will consist of two sides, each side having a different environment, plus a tube that connects the chambers so that the organisms can move from one place to the other. The materials are up to you. Example One group measured the response to moisture over 10 minutes. For their procedure, they set up their behavior chamber so that they had one side moist and one side dry (using paper towels). They transferred 5 isopods to each side of the chamber (total of 10). They counted and recorded the number of animals on each side of the chamber every 30 seconds for ten minutes, using a table similar to the following. Results - Example Time (min:sec) # in Wet # in Dry Other Notes 0:00 0:30 1:00 1:30 Some other suggestions for experimentation may include, but are not limited to… Factor Materials (suggested) Temperature cold pack, warm pack lamps, flashlights, dark construction paper, aluminum Light foil pH low pH (HCl), high pH (NaOH) Substrate (surface) soil, sand, sandpaper, bark, paper, cedar chips, gravel Odor Ammonia Presence of other organisms spider, worm, cricket Food apple, potato, fish food, lunchmeat Discussion Follow the lab report requirements for a full discussion of your findings. P a g e | 54 anana DNA Extraction Introduction Bananas are good sources of DNA. You will break open the cells and then separate the DNA from the remaining cell parts. Next time you eat bananas, perhaps you’ll be thinking about the large amount of DNA that you’re eating. Prediction What do you think the DNA that you extract will look like? Materials Table salt Dishwashing detergent Meat tenderizer Distilled water Isopropyl alcohol Bananas Self-sealing baggie Wooden coffee stirrer Test tube Test tube rack Filter paper Funnel Small scoop Teaspoon scoop 25 ml graduated cylinder Microcentrifuge tube (optional) Methylene blue (optional) Procedures 1. Place a third of a banana into a baggie, press the air out, seal it, and mash for 2 minutes. 2. Add a teaspoon of salt and 20 ml of water to the bananas and repeat the previous step for 1 minute. 3. Pour the mixture through the filtration apparatus, collecting it into a test tube. 4. Add approximately as much dishwashing detergent as filtrate to the test tube. No more than 5 ml are required. The test tube should be about half full after this step. 5. Add a small scoop of meat tenderizer. 6. Mix gently for about 2 minutes by covering and inverting the test tube and also swirling it. Make sure not to be rough or you will break up the DNA molecules. 7. Very slowly add several milliliters of isopropyl alcohol to the extract by pouring it gently down the side of the test tube. The test tube should be about 2/3 full after this step. Do not stir it. 8. Alcohol will be floating on top of the mixture. Wait a few minutes and stringy globs will appear in the alcohol. That is DNA! 9. Dip the wooden stirrer through the alcohol, into the DNA/buffer solution. Gently twirl the stick just below the boundary of the two layers. After a minute of twirling, slowly pull the stick up out of the test tube. 10. Make observations. 11. You may add methylene blue to the rest of the test tube. What does this do? 12. If you wish, the DNA can be saved. Obtain a microcentrifuge tube and add some DNA to it by scraping it into the tube. P a g e | 55 Discussion 1. List 5 things available to you that you might use for a DNA source besides bananas. 2. List 5 things that would not be a DNA source. 3. Why did you use detergent in the procedure? What does it do? 4. Which ingredient do you think had the most effect on the amount of DNA you extracted? Describe an experiment that you could perform to test this. 5. Why is DNA important to living things? 6. Was your prediction correct? Why or why not? P a g e | 56 During the cell cycle, which stage do onion root cells spend the most time in? Objectives Determine the amount of time onion cells spend in each stage of the cell cycle Introduction In some cells, such as human muscle cells and nerve cells, mitosis never occurs. In plants, this is not true. Plant cells continually undergo mitosis. In this investigation, you will identify the various stages of the cell cycle and the stage plant root cells spend the most time undergoing. Materials (per group) Textbook Prepared slide of plant mitosis (onion root tip) Onions Toothpicks Beakers of Water Microscope Safety goggles Metric ruler Ethanol-acetic acid Scalpel Forceps 3-50 ml beakers Glass marking pencil Hydrochloric Acid Clock with second hand 2 glass slides 2 medicine droppers Toluidine blue Probe Paper towels Cover slip Procedure 1. One week to 10 days before the lab, insert toothpicks into an onion bulb and suspend it in a beaker of water so that the bottom of the bulb is under the water. Allow the roots to grow to a length of at least 1 cm. 2. One day before the lab, prepare the fixative with 75 ml of ethanol to every 25 ml of glacial acetic acid. Work only where there is sufficient ventilation, as the fixative is volatile. 3. Cut a 3 mm piece from the tip of each root and place it in a beaker of the prepared fixative. Try to do this around noon because of the occurrence of mitosis peaks at this time (as well as midnight). 4. Try to keep the roots in the fixative for at least 4 hours (but not longer than 48 hours). The fixation will “stop the action” of the dividing cells and preserve them in their current state. The acid will soften the plant cell walls to make the “root tip squash” easier. 5. Label the three 50 ml beakers with the glass marking pencil as follows: fixative, HCl [use 5 M (18%) Hydrochloric Acid], water. Put on your safety goggles and add 10 ml each liquid to the appropriately labeled beaker. CAUTION: Because acids can burn the skin, handle them with care. Place the beaker containing the water aside for now. 6. Using forceps, remove the tips from the fixative and place them in the HCl beaker for 4 minutes. 7. After 4 minutes, use the forceps to remove the root tip from the HCl and place it back into into the fixative for 4 minutes. 8. After 4 minutes, use the forceps to remove the root tips from the fixative and place them on a clean glass slide. Holding the root tips with the forceps, use a scalpel and P a g e | 57 cut off about 1 mm from each root tip. CAUTION: Be careful when using a scalpel. Discard the remainder of the root. 9. Place the glass slide on a paper towel. 10. Place a few drops of toluidine blue on the root tip. Allow the toluidine blue to remain on the root tip for 2 minutes. After 2 minutes, use a paper towel and absorb the excess liquid. 11. Using a clean medicine dropper, place 2 drops of water on the root tip. Cover it with a cover slip. 12. Place a paper towel over the slide, and with the eraser end of the pencil, gently press down on the area covered by the cover slip. This will squash the root tip. CAUTION: Do not press so hard as to break the cover slip or the glass slide. 13. With the microscope, examine the onion root tip under low power to find the cells in various stages of the cell cycle. (Note: If you cannot clearly distinguish the stages of the cell cycle, repeat steps 6 through 12 using another root tip.) 14. After you have located the various stages of the cell cycle under low power, switch to high power. If you have digital access, capture a digital micrograph of cells in each stage of the cell cycle. If not, draw out a cell in each phase. 15. Next, focus on one area of the root tip at low power. You should be able to see the various stages of the cell cycle. Print out a digital micrograph of the image if you are using a computer. 16. Count the total number of cells in each of the 5 phases. Record this information in the appropriate place in Data Table I. Now add up the results for each phase to get the total number of cells in the field of view. 17. Now combine data from the other groups and determine the percentage of time spent in each of the stages of mitosis. To determine the approximate proportion of time a cell spends in each phase of mitosis, divide the number of cells in each phase by the total number of cells in the field of view. To convert each decimal to a percent, multiply by 100. Record this information in the appropriate place in Data Table II. Results Data Table I: Individual Group Data Plant Cell Mitosis: Phase Number of Cells in Each Phase Interphase Prophase Metaphase Anaphase Telophase Total number of cells in field of view P a g e | 58 Data Table II: Class Data Number of Cells in Each Percentage of Cells in Phase Phase Each Phase Interphase Prophase Metaphase Anaphase Telophase Total number of 100% cells investigated **Be sure to attach your digital images. Discussion 1. In which phase of the cell cycle is the most time spent? Why? 2. How much time is spent during mitosis (PMAT)? How does this compare to the total time for the cell cycle? 3. What are plant and animal cells doing when they are not undergoing mitosis? 4. Yeast is capable of undergoing one full cycle in 30 minutes. Using the information that you obtained from your analysis, how long Yeast spends in each phase? Interphase Prophase Metaphase Anaphase Telophase P a g e | 59 Principles of Genetics Introduction Through his careful and detailed studies with garden pea plants, the Austrian monk and scientist Gregor Mendel determined that heredity is controlled by factors that are passed from generation to generation with predictable results. Mendel’s experiments and conclusions led to the formation of several basic genetic principles. The principle of dominance states that some factors (alleles) are dominant, whereas others are recessive. The effects of a dominant allele are seen even if it is present with a contrasting recessive allele. The principle of segregation states that during gamete (sperm and egg) formation, the alleles for a trait separate, so that each gamete has only one of the alleles for that trait. The principle of independent assortment states that as gametes form, the genes for various traits separate independently of one another. In this exercise, you will construct Punnett squares to observe the principles of genetics that were based on Mendel’s work. You will also simulate a dihybrid (two- factor) cross to learn the important role probability plays in the study of genetics. Objective To predict the outcome of a dihybrid cross Materials plastic cup masking tape scissors plastic disks (4) marker Procedures Part A : Constructing Punnett squares for one-factor and two-factor crosses. 1. Examine Figure 1. The capital letter T is used to represent the dominant allele for tallness in the stem length for pea plants. The lower case letter t is used to represent the recessive allele for shortness in stem length for pea plants. Notice the genotypes or gene combinations that result from the parental (P1) generation cross between a female purebred tall (TT) pea plant and a male recessive short (tt) pea plant. All of the offspring that make up the first filial (F1) generation are tall (Tt). Their phenotypes, or visible characteristics, are all tall. P a g e | 60 Purebred tall female x Short male P1 generation Genotype TT x tt Female gametes T T t Male gametes Tt Tt t Tt Tt genotypic ratio = 100% Tt F1 generation phenotypic ratio = 100% tall Figure 1 2. Figure 2 shows the results of a one-factor cross between the offspring from the F1 generation. Observe the genotypic and phenotypic ratios for the second filial generation (F2). When writing such ratios, the numbers for the dominant genotypes or phenotypes come first. In this example, ¼ of the offspring are TT, two fourths or ½ are Tt and ¼ are tt. The genotypic ratio therefore is ¼ : ½ : ¼ or 1 : 2 : 1. Three fourths of the offspring are tall and one fourth are short so the phenotypic ratio is ¾ : ¼ or 3 : 1. Hybrid tall female x Hybrid tall male F1 generation Genotype Tt x Tt Female gametes T t T TT Tt Male gametes t Tt tt genotypic ratio = 1 : 2 : 1 F2 generation phenotypic ration = 3 : 1 Figure 2 P a g e | 61 3. In soybeans, purple flower color is dominant and white flower color is recessive. Let P represent the dominate allele (purple) and p the recessive allele (white). 4. Predict the possible genotypes of the first filial generation (F1) of offspring by completing the Punnett square in Data Table 1. Indicate the genotypic and phenotypic ratios of the F1 generation. 5. In Data Table 2, cross two plants from the F1 generation. Write the appropriate genotypes for this cross on the lines provided. Predict the probable genotypes of this cross by completing the Punnett square in Data Table 2. 6. Indicate the genotypic and phenotypic ratios of the F2 generation in Data Table 2. 7. In corn plants, rough seed shape (R) is dominant over smooth seed shape (r) and yellow seeds (Y) are dominant over white seeds (y). Determine the probable seed shape and color of the F1 generation of offspring whose parents are heterozygous for the traits by completing the Punnett square in Date Table 3. Then, fill in Data Table 4. Observations Data Table 1 Purebred purple female x White male P1 generation = PP x pp Female gametes Male gametes Genotypic ratio = F1 generation Phenotypic ratio = P a g e | 62 Data Table 2 F1 generation = ________ x _________ Female gametes Male gametes Genotypic ratio = F 2 generation Phenotypic ratio = Data Table 3 P1 generation = RrYy x RrYy Female gametes RY Ry rY ry RY Ry Male gametes rY ry P a g e | 63 Data Table 4 Genotype Genotypic Ratio Phenotype Phenotypic ratio RRYY RrYy RrYY RRYy RRyy Rryy rrYY rrYy rryy Part B: Simulating a Two-Factor Cross 1. To simulate the two-factor cross discussed in step 7 of Part A, cut 8 pieces of masking tape just large enough to fit on the plastic disks. Place the tape on both sides of all 4 disks. 2. On one side of two of the disks write R. On the other side of these, disks write r. Repeat this with the other two disks writing Y on one side and y on the other side. 3. Each labeled disk represents the alleles in the heterozygous plant. Tossing the disks together represents the crossing of the heterozygous plants. 4. Place the 4 labeled disks into the plastic cup. Holding one hand over the top of the cup, shake it so that the disks are tossed together in the cup. Then, empty the cup onto the lab table and record the results of the toss by making tally marks in the appropriate spaces of Data Table 5. 5. Toss the disks a total of 48 times, recording the results of each toss in Data Table 5 count the tally marks for each phenotypic combination and record these totals in the appropriate place in the table. P a g e | 64 Data Table 5 Number Number Total Expected Expected Phenotypes Genotypes Toss Results Number For 16 For 48 observed Offspring Offspring RRYY Rough, yellow RrYy seeds RRYy RrYY Rough, white RRyy seeds Rryy Smooth, yellow rrYY seeds rrYy Smooth, white rryy seeds Discussion 1. When two homozygous plants with contrasting traits are crossed, what are the expected genotypes for the offspring? 2. What is the expected genotypic ratio for a one-factor cross of two hybrid organisms? 3. What is the expected phenotypic ratio for a one-factor cross of two hybrid organisms? 4. In your simulated two-factor cross, why might your actual experimental values have been different from the values you expected? 5. Why is it helpful to conduct a large number of trials when simulating genetic crosses? P a g e | 65 Application 1. Why would animal or plant breeders need an understanding of genetics? 2. Is it possible for two organisms to have different phenotypes but the same genotype? Explain your answer. 3. Is it possible for two organisms to have different genotypes but the same phenotype? Explain your answer. 4. How could a guinea pig breeder determine whether a rough-coated guinea pig is homozygous or heterozygous for this trait? 5. In dogs, wire hair is due to a dominate gene W. Two wire-hair dogs were mated and produced a puppy with smooth hair. What were the genotypes of the two parent dogs? 6. Repeat Part B of this activity using a cross between the genotypes RrYy and Rryy. Construct a Punnett square on a separate sheet of paper to represent your expected results and calculate the expected genotypic and phenotypic ratios. Tally the results of your 48 tosses and compare your experimental values with the expected values. P a g e | 66 Applying Mendelian Genetics Meiosis Gamete Production – Demonstrating Crossing-over Objective To understand the principles of gamete production through Mendelian Genetics. Materials scissors, tape, data table, chromosome handouts, and a portrait sheet Procedure 1. Find a partner to work with. Opposite sex partners work the best! 2. You are to determine the phenotype characteristics of an offspring through meiotic gamete production. 3. The table below determines the phenotypes and genotypes for each partner. Each partner will complete that characteristics information for Data Table I. Phenotype Characteristics Skin Color If you have dark hair and dark colored eyes your phenotype is dark skin and your genotype contains both dominant alleles for the gene- SS. If you have either light colored eyes OR light hair color, then your phenotype for skin type is light skin and your genotype is heterozygous-Ss. If you have BOTH light colored eyes and light colored hair, then you possess a white skin color with a recessive genotype of ss. Hair Style If your hair is straight then you are homozygous dominant (HH) and have the straight phenotype. If your hair is wavy, then you are heterozygous (Hh) with wavy hair. If your hair is curly, then you are homozygous recessive and have the genotype hh. Hair Color If your hair is black, then your color is dark and you are homozygous dominant (CC). If your hair color is blonde, then you are homozygous recessive (cc) with light hair. If your hair is somewhat in between, then your phenotype is mixed color and you carry the alleles Cc. Eye Color If your eye color is brown then your genotype is EE and your color is brown. If your color is blue, then you are homozygous recessive and your phenotype is blue. If your color is green, then your genotype is Ee. Body If your weight is above 150 lbs, then your phenotype is round and you carry the Morphology alleles GG. A weight of 125-149 lbs denotes a phenotype of husky and alleles Gg. A weight less than 125 lbs denotes a phenotype of slim and possesses the alleles gg. Sex If you are a male your genotype is XY. Females have the genotype XX. Face Shape Let your partner be the judge. If you have a round face, then you are Homozygous dominant (AA) and have the phenotype round. If you have the phenotype of oval, then your genotype is heterozygous (Aa). If you have the phenotype of square, then your genotype is aa. Arm/ Leg If you are 5’10” or larger, then you have the phenotype tall and possess Length homozygous dominant alleles (TT). If you are 5’4” – 5’9” then you carry the phenotype of medium height and the alleles Tt. A height of 5’3” or less has the phenotype of short and alleles tt. P a g e | 67 4. Each student must complete the lab activity in developing his or her own gametes. 5. The traits for genes 1-4 are found on chromosome pair #1 and the traits for genes 5-8 are found on chromosome pair #2. Write your alleles on the chromosomes as they appear in the chart. 6. Cut out the sister chromatids and temporarily connect the duplicate and the original at centromere. Then, form a tetrad by connecting the homologous pairs together. You should have two tetrads. DO NOT OVERTAPE THROUGHOUT LAB. YOU WILL TAKE THE CHROMOSOMES APART SEVERAL TIMES. Use these chromosomes as you work through the stages of meiosis with the following chart. At the end of the chart you will have created the sex cells needed for reproduction. **Go through meiosis I & II using chart** 7. Complete the sketch with a working model of two chromosomes going through the steps of meiosis I and II found on pages 3 and 4. Use your desktop surface for the working model. At the conclusion of your sketch and model, you will have 4 gametes (sperm or egg) on your desktop. 8. Using the directions from the meiosis chart, mate the male and female sex cells together. Write the alleles of the offspring that will be produced in Data Table II. 9. Draw your child that will be produced from your initial characteristics. Be sure to use colored pencils and write its name on the portrait sheet! 10. You must turn in the data tables and pictures. P a g e | 68 Meiosis I [As you sketch your model in the circles below, make a working model on your desktop.] Replicate your 2 chromosomes using the base chromosome pairs. Make sure that the Interphase replicated set is identical. There are two models working, dad and mom. Each student does their own! Pair up the homologous chromosomes at the centromere to form a tetrad . This is called synapsis. Then illustrate crossing over. Do Prophase I this by cutting off either 1 or 2 genes on the crossed over section of each chromosome. Next, re-tape the genes on the homologous chromosome. Metaphase I Line up the crossed over chromosomes on the metaphase plate in the center of your desk. Anaphase I Separate the tetrads, showing the tetrads being pulled to the opposite ends of your cell (desk). Telophase I Now you should have 2 cells, both having 1 set of chromosomes for chromosome 1 and 2. P a g e | 69 Meiosis II Prophase II During this stage, there is no noticeable activity. Leave your chromosomes alone here. In both cells the chromosomes line up Metaphase II along the metaphase plate. Show this on your desk. Keep in mind that you are still working with 2 different cells. In each cell, split the sister chromatids and show them moving to opposite Anaphase II poles. Make sure that each pole in each cell contains one chromosome 1 and one chromosome 2. Telophase II Line the 4 gametes (sex cells) up along your desk. The third one from the left will be used for reproduction with your partner’s gamete. Discussion 1. What is the difference between a chromatid and a tetrad? 2. What is synapsis? What is the only stage that synapsis occurs? 3. What is the significant difference between the development of the sperm and the egg? 4. What are polar bodies? P a g e | 70 Data Table I: Characteristics of Parents Mom Mom Genotype Dad Phenotype Dad Genotype Genes Phenotype 1. Skin Type 2. Hair Style 3. Hair Color 4. Eye Color 5. Body Morphology 6. Sex 7. Face Shape 8. Arm/ Leg length Data Table II: Characteristics of Child Allele from Allele from Genotype of Phenotype of Genes Mother Father Child Child 1. Skin Type 2. Hair Style 3. Hair Color 4. Eye Color 5. Body Morphology 6. Sex 7. Face Shape 8. Arm/ Leg length P a g e | 71 How Can Blood Typing Be Used To Determine Parent- Offspring Relationships? Objectives Simulate blood typing Use blood typing to solve a parent-offspring relationship case Materials 2 Spot Plates Test Tube Brush Detergent Dropper Bottles with: Recipient Type A Recipient Type B Recipient Type AB Donor Blood Types: A, B, AB & O Unknown Children and Parent Blood Samples Introduction Understanding blood types is important in the transfusion of blood. Fill out this chart based on what you know about blood types and antigens. Remember, a person can only receive antigens that they are familiar with. Blood Types Antigens in Red Can Donate To… Can Receive Blood Cells From… A B AB O 1. Which blood type is known as the universal donor? Why? 2. Which blood type is known as the universal acceptor? Why? P a g e | 72 The ABO blood groups are determined from a single gene with three possible alleles: IA, IB, or i. Since IA and IB are both expressed when they occur together, they are said to be co-dominant. IA and IB are both dominant over i. Fill in the following chart to illustrate the genotypes of individuals with each of the four blood types. Phenotype Genotype A _____ _____ or _____ _____ B _____ _____ or _____ _____ AB _____ _____ O _____ _____ In addition to the ABO antigens, there is another antigen on the red blood cells that determines the success or failure of transfusions. It is the Rh antigen named after the rhesus monkey in which the antigen was first discovered. People who have the antigen on their red blood cells are Rh positive (Rh+). People without the antigen are Rh negative (Rh-). In blood banks the ABO and Rh groups are often expressed together in symbols such as AB- or O+. The gene for the Rh antigen displays simple dominance, where + is dominant over -. Fill in this chart to help illustrate the genotypes of different Rh blood types. Denote each allele as either Rh+ or Rh-. Phenotypes Genotypes Rh Positive _____ _____ OR _____ _____ Rh Negative _____ _____ Use your knowledge of blood types and genetics to solve the following problem. 1. A man with blood type AB marries a woman with type O blood. What blood types would you expect to see in their children and in what percentage? Show your work. P a g e | 73 Procedures Part I: Universal Donors and Recipients – Demonstrating Blood Type Tests 1. Set up the spot plates as instructed. The first row of four people will all have type-A blood, the second row of four people will all have type-B blood, the third row will have type-AB, and the fourth will have type-O blood. Place the spot plate on the chart on this page to aid you in placing blood in each well. Using the dropper bottles, place 3 drops of type-A (recipient) blood in the first column for the A-recipients. These represent the recipients with type-A blood. 2. Now fill the other columns with 3 drops of the correct recipient blood (A, B, AB, or O). 3. Now we are going to perform a blood transfusion to each of the 20 different people. First add 3 drops of type-A donor blood to the first individuals in rows #1-4. Add 3 drops of type-B donor blood to the 2nd individuals in rows #1-4, and so on. See the chart for assistance. 4. On the chart below write draw a “smiley” face to show a successful transfusion of blood. This shows that the recipients and donors are compatible. Draw an unhappy face to show that the transfusion was not successful and the recipient did not survive. 5. Discard solutions in the sink, wash spot plate with detergent and test tube brush. Recipients Blood Type A B AB O A B Donors AB O Part II: Whose baby is it? On a dark, stormy night in Pittsburgh, four newborn babies were being transferred to the nursery in a prestigious University Hospital when a power outage occurred. In the ensuing confusion, one of the new residents inadvertently mixed up the four newborns. Although embarrassed by the mistake, the responsible resident reported the situation to the chief resident. You are the lab technician, which received the blood samples taken from the parents and newborns. Through analysis of the parents and newborn babies’ red blood cell antigens, it is now up to you to determine the parents of each of the newborns. The Rh factor is provided to help you. Good luck!! P a g e | 74 1. Obtain samples from each of the children and parents (3-drops/ well). Use the data table so you know what goes into each well. 2. At your seat, determine the blood type of the children and parents using additional wells on your spot plates. Use the dropper bottles of type A, B, and AB recipient blood types to determine the presence or absence of “A” and “B” antigens. (The Rh factors are already recorded). Record the blood types in the table below. 3. Discard solutions in the sink and wash the spot plates with detergent and test tube brush. Human Blood Groups Data Table Recipients Type A Type B Type AB Type O Blood Type Baby 1 Baby 2 Baby 3 Baby 4 Mr. Brown Donors Mrs. Brown Mr. Gray Mrs. Gray Mr. Smith Mrs. Smith Mr. Jones Mrs. Jones P a g e | 75 4. Using the phenotypes of the parents (from the blood samples examined), determine the possible outcomes for the babies’ alleles. Remember, each parent has two alleles. For type A and type B parents, assume that they carry the recessive allele. Possible Offspring Genotypes (Show all work for the possible outcomes of the following parents. You may use a Punnett Square to help) Mr. & Mrs. Brown Mr. & Mrs. Smith Mr. & Mrs. Jones Mr. & Mrs. Gray Fill in the following table using the blood types determined through testing. Baby Blood Types Baby Blood Type Rh Factor (Including Rh) 1 + 2 + 3 - 4 + P a g e | 76 Parents Offspring Name Rh Factor Blood Type Possible Rh Possible Blood Factor(s) Type(s) Mr. Brown + Mrs. Brown - Mr. Gray + Mrs. Gray - Mr. Smith - Mrs. Smith - Mr. Jones + Mrs. Jones - Once you have determined all of the possible outcomes, determine which child belongs to which set of parents. Record the parents below. Parent-Offspring Relationships Child Parents 1 2 3 4 Application 1. Suppose 2 newborn babies were accidentally mixed up in the hospital, and there was a question of which baby belonged to which of the parents. From the following blood types, determine which baby belongs to which parents. Mrs. Schrader Type B+ Mr. Schrader Type AB- Mrs. Hathy Type B+ Mr. Hathy Type B+ Baby 1 Type O+ Baby 2 Type A+ 2. What are the genotypes of each of the six people listed above? P a g e | 77 3. Complete the family tree below and give the genotypes of the following individuals. A B A B AB A A O A A O A A A A O B B A B B O 4. What are the possible blood types of children in the following families? Type-A+ mother, type-A- father Type-A- mother, type-O- father Type-B- mother, type-AB- father Type-AB+ mother, type-AB- father Type-A+ mother, type-B+ father Discussion 1. Write a paragraph explaining the importance of blood typing. In what ways is blood typing useful? What are the limitations of blood typing? What is your blood type? P a g e | 78 Karyotyping Introduction This lab is will help us understand the differences among the autosomes (chromosomes #1-22) and the sex chromosomes (Chromosome #23). With the help of the human genome project, scientists have mapped the location of many genes in human chromosomes. The locations of important mapped genes are shown on your chromosomes. Objectives Combine your chromosomes with another member of the class and determine the genetic make-up of the child that should be produced Use mapped genes to determine the genotype and phenotype of your offspring Procedure 1. Find your lab partner by locating the person with the same numbered envelope. The envelope that you received contains paternal (male) or maternal (female) chromosomes. If your chromosomes are pink, you are the mother. If your chromosomes are blue, you are the father. 2. Once you and your partner find each other, spread out the contents of each of your envelopes. Mix up the chromosomes then try to match them up together by number, size, banding, gene location, etc. Occasionally, there may be a missing or extra chromosome. In this instance, you have a genetic disorder. 3. After you finish karyotyping your chromosomes, you should notice that your baby probably has one type of genetic disease. 4. Use this karyotype to answer the following questions. Then put the chromosomes back in their respective envelopes. Be sure to inform the teacher of any chromosomes that are missing. Results COUPLE NUMBER SEX OF CHILD GENETIC DISEASE WHICH DISEASE IS HE/SHE A CARRIER OF? BLOOD TYPE HAIR COLOR EYE COLOR P a g e | 79 Discussion 1. How many chromosomes are found in human sperm? 2. How many chromosomes are found in human ova? 3. How many chromosomes are found in each cell of a newborn child? 4. How many chromosomes are found in your created child from this lab? 5. What form of cell division creates gametes (sperm and egg)? 6. Which form of cell division creates identical diploid cells? 7. What is the relative size of an X-chromosome to a Y-chromosome? 8. What genetic disorder(s) did the sperm carry? 9. Which genetic disorder(s) did the egg carry? 10. What genetic disorder has your baby inherited? Going Further After you complete the karyotyping lab you will discover that your baby has inherited a combination of genes or chromosomes that result in a genetic disease. Your assignment is to find information on your baby’s genetic disease, including: Symptoms, onset, frequency, mode of inheritance, chromosome and locus, treatment, and lifespan. P a g e | 80 Radioactive Decay Introduction It was not until the end of the 1800s that scientists found a method for determining the actual age of rocks and minerals. They found that a given mass of a radioactive element decays, or changes to other elements, by giving off particles and energy at a constant measurable rate. Scientists also found that for each different radioactive element the rate of change was fixed and not at all affected by such things as the pressure or temperature of the surrounding environment. The decay process is so regular that it can literally be used to determine the passage of time like the ticking of a clock. In this activity you will use a mathematical model to study the process of radioactive decay in order to help understand how it can be used to determine the age of ancient earth materials. For this activity, it will be helpful to remember that the term half-life refers to the time required for half of the atoms of a given mass of substance to decay. Objective To gain an understanding of what is meant by a half-life. Materials cardboard box with lid plastic cup with corn kernels graph paper Procedure 1. Count out exactly 100 kernels of corn and place them in the box provided. Note that the insides of the box have been numbered 1 - 4. 2. Place the lid on the box and give it one hard shake and place it on the lab table. 3. Carefully open the box and remove all the corn kernels that have the small end pointing toward the side numbered 1. Count them and subtract that number from 100. This will be the number of kernels remaining in the box. Record this number in the proper place in the data table. Do not return the kernels you removed from the box. 4. Replace the lid and give the box another shake and once more remove all the kernels pointing toward side 1. Count them and subtract this number from those remaining from the previous shake. Record this new figure in the data table. 5. Repeat this process until all of the corn kernels have been removed from the box. 6. Return all 100 pieces of corn to the box, cover it, and repeat the above procedure; except this time, after each shake, remove all the kernels pointing towards both sides 1 and 2. Calculate the corn remaining in the box and record your data. 7. Continue this procedure until all the corn has been removed from the box. 8. Finally, return all 100 pieces of corn to the box and repeat the entire procedure a third time, except after each shake remove all those kernels pointing toward sides 1, 2 and 3. Continue this process again until all kernels have been removed. Make sure to record your data in the proper place in the data table. P a g e | 81 Results Shake # Substance A Substance B Substance C Corn Remaining Corn Remaining Corn Remaining Side 1 Test Sides 1,2 Test Sides 1,2,3 Test 0 100 100 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 **Using your data, construct a graph with number of corn kernels remaining on the Y-axis and number of shakes on the X-axis. Your graph should have 3 lines, each representing a different substance. P a g e | 82 Discussion 1. Estimate the half-life for each set of data (approx. # of shakes to reduce kernel number by one half)? 2. Are the half-lives the same or different for each substance? Explain. 3. Radioactive C14 has a half life of 5,770 years, decaying into N14. Assuming living things take in C14 while alive, you can compare it to how much N14 exists in a sample. If a fossil is found to consist of 1/8 the amount of C14 it would be expected to have, how old would it be? Show your work. P a g e | 83 Examining the Fossil Record Objectives Analyze characteristics of fossils Compare placement of fossils and determine relative ages Develop a model evolutionary tree based on the morphology and age of fossils Background Fossils are traces of organisms that lived in the past. When fossils are found, they are analyzed to determine the age of the fossil. The absolute age of the fossil can be determined though radiometric dating and determining the layer of rock in which the fossil was found. Older layers are found deeper within the earth than newer layers. The age and morphologies (appearances) of fossils can be used to place fossils in sequences that often show patterns of changes that have occurred over time. This relationship can be depicted in an evolutionary tree, also known as a phylogenetic tree. There are two major hypotheses on how evolution takes place: gradualism and punctuated equilibrium. Gradualism suggests that organisms evolve through a process of slow and constant change. For instance, an organism that shows a fossil record of gradually increased size in small steps, or an organism that shows a gradual loss of a structure. Punctuated equilibrium suggests that species evolve very rapidly and then stay the same for a large period of time. This rapid change is attributed to a mutation in a few essential genes. The sudden appearance of new structures could be explained by punctuated equilibrium. Speciation The fossil record cannot accurately determine when one species becomes another species. However, two hypotheses regarding speciation also exist. Phyletic speciation suggests that abrupt mutations in a few regulatory genes occur after a species has existed for a long period of time. This mutation results in the entire species shifting to a new species. Phyletic speciation would also relate to the Punctuated Equilibrium hypothesis regarding evolution. Divergent speciation suggests that a gradual accumulation of small genetic changes results in subpopulation of a species that eventually accumulate so many changes that the subpopulations become different species. This hypothesis would coincide with the gradualism model of evolution. Most evolutionary biologists accept that a combination of the two models has affected the evolution of species over time. P a g e | 84 Procedure 1. The diagram you are creating requires a large space. To create your workspace, tape together 6 sheets of standard sized paper. Use a ruler to draw the following chart on your workspace Time Period Began (years ago) Fossils ( 2 inches wide) ( 2 inches wide) (7 inches wide) Wyomingion 995,000 (6 inches tall) (oldest) Ohioian 745, 000 (6 inches tall) Nevadian 545,000 (6 inches tall) Texian 445,000 (6 inches tall) Oregonian 395,000 (6 inches tall) Coloradian 320,000 (6 inches tall) Montanian 170,000 (6 inches tall) Californian 80,000 (6 inches tall) Idahoan (present) 30,000 (6 inches tall) 2. The groups of "fossils" you will work with are fictitious animals. Each fossil on your sheet is marked with a time period. Cut out each fossil and make sure you include the time period marked below it. 3. Arrange the fossils by age. On your data chart, place each fossil next to the period from which the fossil came from. The term "upper" means more recent and should be placed lower than the low. The term "lower" means an earlier time period, fossils from a "lower" time period should be place toward the older time periods. In each fossil column, you may have 3 specimens, one from the main time period, one from the upper, and one from P a g e | 85 the lower. Not all fossils are represented, illustrating the incompleteness of any fossil record. 4. While keeping the fossils in the proper age order, arrange them by morphology (appearance). To help you understand the morphology of the specimen, view the diagram to the right. Arrange the fossils using the following steps: a. Center the oldest fossil at the top of the fossil column (toward the oldest layer) b. Throughout the chart, those fossils that appear to be the same (or close to the same) as the fossils preceding them should be placed in a vertical line c. During a certain period, the fossils will split into two branches. In other words, one fossil from that period will show one type of change, and another fossil will show a different change. When this happens, place the fossils side by side in the appropriate time period. From this point on you will have two lineages. 5. Once all the fossils have been placed correctly according to time and morphology, tape or glue the fossils in place. Discussion 1. Give a brief description of the evolutionary changes that occurred in the organism. 2. During which time period(s) did the fossils differentiate into two branches? 3. Explain how the chart illustrates both punctuated equilibrium and gradualism. Use specific fossils from the chart to support your answer. 4. Making the assumption that each fossil represents a separate species. Explain how the chart illustrates divergent and phyletic speciation. Use specific fossils from the chart to support your answer. P a g e | 86 5. Define the following terms: morphology – fossil – phylogenetic tree – 6. Examine the fossil that was unearthed in a museum, apparently the labels and other information were lost. Based on the fossil record, this fossil is likely from what period? 7. There were two different species found during the Upper Montantian period. When did these species share a common ancestor? How do you know? P a g e | 87 8. What environmental conditions do you suppose led to each of the different lineages? How is each lineage adapted for their environment? 9. Which of the two major species that arose from the parent species was more successful? How do you know? 10. For each of the "blanks" on your fossil record, make a sketch of what the animal would look like. Draw them directly on your fossil record. Also draw in the environment in the background for where you would have expected the organisms to live. P a g e | 88 Fossils P a g e | 89 Natural Selection: The driving force of evolutionary change Big Ideas Favorable traits increase the “fitness” an organism. An organism’s “fitness” involves: o The likelihood that it will survive, and o The amount of offspring that it is likely to produce. Over time, as more “fit” members of a population survive more and reproduce more, populations will become more and more “fit” overall (i.e. the “fittest” traits will become more and more prevalent over time). Related Ideas Communities have carrying capacities, meaning that they can only support a limited amount of organisms. Competition for resources occurs between individuals of a population, where more fit individuals are more likely to survive. Competition for resources occurs between populations of a community, where more fit populations are likely to survive. New traits randomly occur. They do not occur on an “as needed” basis. Changes in the environment can change the fitness of organisms, and therefore alter the path of evolutionary change. Materials (per group) 1 penny Handful of Styrofoam 1 paper bag Plastic wrap 1 plastic bag Paper towels 1 straw (cut into 2 halves) Scotch Tape Handful of cotton balls Other materials (per class) Electric fan (large and powerful if possible, elevated about 1-2 meters off the ground if possible) 10-m tape measure As much unobstructed space as possible for a “testing area”, preferably with a hard floor so the seeds can “slide” a little too. Optional: Feathers, Filter Paper, Rubber Bands, Thread/Needle P a g e | 90 Introduction As a team of field ecologists, you have been asked to conduct a study of population change over time. More specifically, you have been asked to accumulate data about how the newly discovered plant species Plantus pennius adapts to its environment over time. This is a plant whose seeds look like pennies, and their primary means of seed dispersal is by wind. Upon discovering the initial population, you notice that the seeds actually show enormous variation. They all contain the part that looks like a penny because this is the actual seed that will germinate into a new plant. They seem to come in a vast variety of casings, though. This discovery leads you to form a hypothesis that the type of seed a particular plant produces will be directly related to the likelihood that the plant will survive and reproduce. Phase I: Discovering the initial population Each lab group will represent a different seed (and therefore a different plant) in each of two generations. The first generation will be the initially discovered population. The second generation will represent the population after one round of “adaptation” to the environment. Table 1 represents the traits that we will use, the variations of those traits, and the abbreviations that we will use for them. Table 1: Alleles and abbreviations Trait # Dominant allele Recessive allele 1 Paper bag casing P Plastic bag casing p 2 Seed taped to inside wall of bag. T Seed freely placed in bag t 3 Bag casing is closed L Bag is open-ended l Bag shape internally supported with Bag shape contains no straw 4 1 straw half per allele R supports r 5 Bag contains cotton balls F Bag contains no cotton balls f 6 Bag contains Styrofoam M Bag contains no Styrofoam m 7 “Parachute” attached H No parachute h 8 Plastic wrap parachute B Paper towel parachute b P a g e | 91 Now discover what the traits of your initial individual plant seed will be. Remember, every organism will have two alleles for every trait, which can be the same or different. 1. Determine the alleles for the first trait (paper bag casing or plastic bag casing) by rolling the die twice (once for each allele). Document the number of each roll in Table 3. Repeat for each trait. 2. Determine the alleles your individual will have for each trait. Refer to Table 2 below to determine which numbers rolled represent which alleles. Then document the genotypes in Table 3. Table 2: Rules for determining alleles Trait # Roll Allele Roll allele 1 1, 2, 3 P 4, 5, 6 p 2 1, 2, 3, 4 T 5, 6 t 3 1, 2, 3 L 4, 5, 6 l 4 1, 2, 3, 4 R 5, 6 r 5 1, 2 F 3, 4, 5, 6 f 6 1, 2, 3 M 4, 5, 6 m 7 1, 2 H 3, 4, 5, 6 h 8 1, 2, 3 B 4, 5, 6 b 3. Based on the genotype, determine the phenotype of your individual seed for each trait, and document it in Table 3. Refer to Table 1 as a reminder of what the alleles mean. 4. Combine all of the phenotypes you discovered, and build a model of your initial seed using the materials available to you. Table 3: Traits of the initial individual Numbers rolled Genotype Trait Phenotype Roll 1 Roll 2 Allele 1 Allele 2 1. Paper or plastic? 2. Taped to inside? 3. Closed or open? 4. Straw supports? 5. Cotton? 6. Styrofoam? 7. Parachute? 8. plastic or paper? P a g e | 92 Phase 2: Determining which individuals are most fit Now we are going to determine which seeds in the population are the “fittest” ones. We will simulate seed dispersal by wind by letting our seeds go in front of a fan, and we will claim that the “fittest” seeds in our population are the ones capable of dispersing the farthest. Before we start, let’s form some hypotheses. Document in Table 4 which traits you believe might contribute to making seeds most “fit” (i.e. document which traits you believe will be better for the seeds). Place check marks in the appropriate boxes. Table 4: Fitness hypotheses for each trait Dominant phenotype Recessive phenotype Paper bag casing Plastic bag casing Seed taped to inside wall of bag Seed freely placed in bag Bag casing is closed Bag is open-ended Bag shape internally supported with 1 Bag shape contains no straw supports straw half per allele Bag contains cotton Bag contains no cotton Bag contains Styrofoam Bag contains no Styrofoam “Parachute” attached No parachute Plastic wrap parachute Paper towel parachute Now we will run our first simulation of wind seed dispersal. 1. Lay out the measuring tape in front of the fan so that it measures the distance away. 2. Release your seed in front of the fan (which should be elevated and set to its highest power). 3. Measure the distance that your penny travels to the nearest millimeter. Remember, the actual penny is the important part, so your final measurement should represent the distance that the penny travels. If time permits, you may take three trials, and average them. Record your group’s data in Table 5. P a g e | 93 Table 5: My seed’s dispersal distance data Trial 1 distance (m) Trial 2 distance (m) Trial 3 distance Average distance (m) (m) Now record your own seed’s average distance, as well as the average distances for all other groups in Table 6. When all groups have completed their simulations, rank their fitnesses, with #1 being the best. Finally, average all of the averages in the table, and record this as the average population dispersal distance below Table 6. Table 6: First generation dispersal distances: Average dispersal Group members Fitness rank distance (meters) My group Average population dispersal distance: P a g e | 94 Phase 3: Passing on the traits Now that we have data that ranks the seeds in order of fitness, here are the rules about which seeds survived, which seeds flourished, and which seeds met a very sad fate. Top 3 seeds: Your plants are of excellent fitness. Twice as many of your plants survive as the “average” plant. The bottom 3 groups (whose plants did not survive) will assume responsibility for your duplicates for reproduction purposes. Your original plant will reproduce, so document your initial genotypes into the first two columns of Table 7 (“My plant”). See Table 3 as a reminder of your initial genotypes. Bottom 3 seeds: Your seeds are not very fit at all. None of your plants survived. You have been replaced by one of the top 3, and you will assume responsibility for one of the top 3 seeds for reproduction purposes. Last place Assume responsibility for the extra “first place” seed, which is now your plant. Document the genotypes of your new plant into the first two columns of Table 7 (“My plant”). 2nd to last place Assume responsibility for the extra “second place” seed, which is now your plant. Document the genotypes of your new plant into the first two columns of Table 7 (“My plant”). 3rd to last place Assume responsibility for the extra “third place” seed, which is now your plant. Document the genotypes of your new plant into the first two columns of Table 7 (“My plant”). All other seeds: Your seeds are of average fitness, and your numbers have remained stable. Your original plant will reproduce, so document your initial genotypes into the first two columns of Table 7 (“My plant”). See table 3 as a reminder of your initial genotypes. P a g e | 95 Now that we all have plants that will reproduce for the next generation, all of our plants must be crossed with another. 1. Your group should pick another lab group at random, and cross your plants. 2. Document the genotypes of the plant with which you are crossing yours in columns 3 and 4 of Table 7 (“Crossed with”). 3. Your pair of plants will produce two unique offspring, so separate yourselves and complete the following reproduction rules on your own in Table 7. a. Allele 1 of your offspring will come from your plant (“My plant” column). It will either be allele 1 or allele 2 from your plant (“My plant”). Roll the die. If it is odd, allele 1 of your offspring will be allele 1 from the parent. If it is even, allele 1 of the offspring will be allele 2 from the parent. b. Allele 2 of your offspring will come from the other parent (the “Crossed with” column). Repeat step (a) to determine which allele from the other parent will become allele 2 of the offspring. c. Repeat steps (a) and (b) for the remaining traits. d. Document the phenotypes of the offspring. 4. Now, build a model of your offspring. Each group will do this since there are 2 unique offspring per pair of mating plants. Table 7: Reproduction chart My plant Crossed with Offspring Trait # Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Phenotype 1 2 3 4 5 6 7 8 P a g e | 96 Phase 4: The next generation Now, we are going to test the seeds of the next generation of Plantus pennius plants. Repeat all testing procedures from Phase 2 of this investigation, and complete the charts below. Table 8: My offspring’s dispersal distance data Trial 1 distance (m) Trial 2 distance (m) Trial 3 distance Average distance (m) (m) Table 9: Next generation dispersal distances: Average dispersal Group members Fitness rank distance (meters) My group Average population dispersal distance: P a g e | 97 Phase 5: Tallying the data Now, we are going to summarize the data that compares the fitness of Plantus pennius plants to the possible phenotypes and alleles. Mingle as a class, tally the total number of alleles and phenotypes in the first and second generations, and document them in Table 10. Everyone should refer to Table 3 for generation 1 data and Table 7 for generation 2 data. Table 10: Dominant allele and phenotype tallies for the first and second generation population. Alleles Phenotypes Traits Generation Generation Generation Generation 1 2 1 2 1 Paper bag casing P 2 Seed taped to inside T 3 Bag casing is closed L 4 Internal straw supports R 5 Bag contains cotton F 6 Bag contains Styrofoam M 7 “Parachute” attached H 8 Plastic wrap parachute B Table 11: Recessive allele and phenotype tallies for the first and second generation population. Alleles Phenotypes Traits Generation Generation Generation Generation 1 2 1 2 1 Plastic bag casing p 2 Seed freely placed in bag t 3 Bag is open-ended l 4 No internal supports r 5 No cotton f 6 No Styrofoam m 7 No parachute h 8 Paper towel parachute b P a g e | 98 Phase 6: Analysis 1. List two reasons why seeds capable of dispersing long distances could be considered more “fit” than seeds incapable of dispersing long distances. 2. Think of two other types of traits a plant seed could have that would qualify it as more fit than a plain naked seed that simply drops to the ground. Explain. 3. Refer back to table 1 that summarizes the dominant and recessive traits. Apply your knowledge about what makes a trait “dominant” and what makes a trait “recessive.” Choose any two of these traits, and explain why (on a molecular level) these traits might be dominant. P a g e | 99 4. Did the population become more fit? How does your data support your conclusion? What do you believe is the ultimate fate of the Plantus pennius population in this community? 5. Assuming this population of Plantus pennius survives far into the future, what would you expect the future of this population to look like (as far as the seeds are concerned)? Hint: Which would you say would be the “top 5 fittest” alleles? 6. Look at Tables 10 and 11. Why are the two boxes crossed out in each chart as though they do not apply? What type of inheritance pattern does this represent? Hint: refer to Table 1. P a g e | 100 7. We focused on dispersal distance alone when analyzing the fitness level of these Plantus pennius plants. If over time this environment became almost totally absent of wind, though, this would change things drastically! Name two large- scale types of environmental change that could change the evolutionary direction of this population. Also describe what the fittest seeds might look like in these two situations. 8. Look back at Table 2. Notice that dominant alleles were not always associated with more possibilities of rolling the die. How do you explain this, and how does it relate to real life? 9. Is this lab more consistent with Lamarck’s or Darwin’s ideas about how life changes (evolves) over time? P a g e | 101 Animal Classification and Diversity Using the Animal Diversity Web: http://animaldiversity.ummz.umich.edu/site/index.html Choose a class of animals (ex. Mammalia, Reptilia, Insecta) to research. Within this class, you must classify 10 distinct species. Use the taxonomy of each of these ten different species to develop a phylogenetic tree of the species. o Explain how the taxonomy of the organism helped you to develop a branching pattern between the species. Once you have completed the tree, choose three of the species. Use a search engine on the internet to research the similarities and differences between the three organisms. Key points can include physical characteristics, behavior, habitat, and role in the food web. o Explain which two of the three organisms are most closely related and what makes the third different from the other two. P a g e | 102 Squid Dissection Living organisms have two main goals: To strive for their own survival as individuals. They do this by: o Responding to their environment. o Obtaining, processing, and disposing energy. o Maintaining a stable internal environment a (process called homeostasis). To strive for the survival of the species. They do this by o Adapting to their environment over time (a process called evolution). o Reproducing. As you dissect the squid, freely inspect the external and internal anatomy. Your goal is to identify internal and external features of the squid as falling under one of the above categories. There is substantial overlap between the categories above! For example, teeth can fall under getting energy if it is used for digesting food, and for responding to the environment in cases where they are used purely for self defense. Feel free to use any resources available to assist you in completing the requirements: Requirements: Physically locate 12 anatomical features of your squid. o At least 8 of your features must be internal. o At least 2 of your features must be external. Describe how they fit under one of the 5 categories (response, energy, homeostasis, evolution, or reproduction): o You must have at least 1 feature describing each category. Note your ideas in the following chart. When the above requirements are complete, notify your teacher. Each lab partner will verbally explain your group’s conclusions. o Each partner will be responsible for explaining an appropriate number of conclusions based on the size of the group. o With a dissecting probe, physically point out the features as you explain them to your teacher. (As you do this, I will initial your charts.) Obtain initials from your teacher and submit your lab handout. P a g e | 103 6. 5. 4. 3. 2. 1. Anatomical feature Internal (at least 8) External (at least 2) Response (at least 1) Energy (at least 1) Homeostasis (at least 1) Adaptation (at least 1) Reproduction (at least 1) Description of how the feature relates to the category. P a g e | 104 12. 11. 10. 9. 8. 7. Anatomical feature Internal (at least 8) External (at least 2) Response (at least 1) Energy (at least 1) Homeostasis (at least 1) Adaptation (at least 1) Reproduction (at least 1) Description of how the feature relates to the category. P a g e | 105 Frog Dissection Living organisms have two main goals: To strive for their own survival as individuals. They do this by: o Responding to their environment. o Obtaining, processing, and disposing energy. o Maintaining a stable internal environment a (process called homeostasis). To strive for the survival of the species. They do this by o Adapting to their environment over time (a process called evolution). o Reproducing. As you dissect the frog, freely inspect the external and internal anatomy. Your goal is to identify internal and external features of the frog as falling under one of the above categories. Keep in mind that there is substantial overlap between the categories above. For example, teeth can fall under getting energy if it is used for digesting food, and for responding to the environment in cases where they are used purely for self defense. Feel free to use any resources available to assist you in completing the requirements: Requirements: Physically locate 12 anatomical features of your frog. o At least 8 of your features must be internal. o At least 2 of your features must be external. Describe how they fit under one of the 5 categories (response, energy, homeostasis, evolution, or reproduction): o You must have at least 1 feature describing each category. Note your ideas in the following chart. When the above requirements are complete, notify your teacher. Each lab partner will verbally explain your group’s conclusions. o Each partner will be responsible for explaining an appropriate number of conclusions based on the size of the group. o With a dissecting probe, physically point out the features as you explain them to your teacher. (As you do this, I will initial your charts.) Obtain initials from your teacher and submit your lab handout. P a g e | 106 6. 5. 4. 3. 2. 1. Anatomical feature Internal (at least 8) External (at least 2) Response (at least 1) Energy (at least 1) Homeostasis (at least 1) Adaptation (at least 1) Reproduction (at least 1) Description of how the feature relates to the category. P a g e | 107 12. 11. 10. 9. 8. 7. Anatomical feature Internal (at least 8) External (at least 2) Response (at least 1) Energy (at least 1) Homeostasis (at least 1) Adaptation (at least 1) Reproduction (at least 1) Description of how the feature relates to the category.