BI102 Lab Packet

Transcription

1 BI102 Lab Packet Required for ALL sections of BI102 (Including On-line Classes) Bring the ENTIRE manual to the first day of lab! 1

2 BIOLOGY LAB SAFETY REGULATIONS & STUDENT RESPONSIBILITIES Read these carefully. You are responsible for them! 1. Science labs are inherently dangerous due to chemicals present. Eating and drinking are prohibited in the labs. 2. In case of injury, get the instructor, send someone for the instructor, or call for help immediately. 3. Know where the fire extinguisher is. In case of fire in the lab, use common sense and play it safe. If it s your clothes on fire, yell "FIRE" and roll on the floor or use a coat to smother flames. Chemical fires are dangerous. Water will often only make the situation worse. If you see abundant flames or smell smoke from and unknown source, you should call out "FIRE" and calmly, but quickly, evacuate the building, insisting others leave as well. 4. If you spill chemicals on you or your clothes, rinse the area immediately with running water. If you splash chemicals in your eyes, go immediately to the eye wash station, turn on cold water, remove the red caps and lean down so that the water bubbles into your eyes. Keep rinsing while holding your eyes open and send someone else to get the instructor! 5. Protect your eyes. Handle all chemical, including stains, below eye level. Wear protective goggles or glasses whenever you work with chemicals or microbes. 6. Wash your hands with soap and water whenever leaving the lab for a break or at the end of class. 7. Wash your work area and spray it down with the disinfectant provided before each lab begins. 8. Closed-toed shoes must be won at all times in the lab. Long pants are preferred, even in warm weather, to protect legs. Old clothes without baggy sleeves are recommended. 9. Keep the lab counter uncluttered. Stow extra books and coats on the back counter if available. 10. If you have long hair, tie it back to keep it out of your eyes and the chemicals. 11. Clean-up is your responsibility. Wash down your work area to remove spilled chemicals at the end of lab. Wash all glassware and dry it, including slides and cover slips. Sweep up broken glass and deposit it in the special cardboard container provided (not in the trash cans). Clean out the sink if you have used it. 12. Know what you are working with at all times and be prepared should something go wrong. Read the lab in advance and precisely follow all instructions. 2

3 Biotechnology Project Summary Biotechnology is a new and rapidly growing field that uses the concepts of biology to solve the problems of the modern world. The techniques used in this field save us time and money, improve our health and, in some cases protect the planet. As we become more and more reliant on the various forms of biotechnology, we become more and more aware of the ethical and environmental implications of using these technologies. As a result, biotechnology is often a source of controversy and many people express a desire to regulate or even prohibit these techniques. The goal of this project is to introduce you to several different types of biotechnology. The project will involve researching both the technique itself and any controversy surrounding its use. Based on what you learn during your research, you group will propose some level of regulation for your technology and develop two political campaign ads. One ad will support the regulation of the technology and the other ad will oppose regulation. In the end, you should be able to provide an informed opinion about the use of biotechnology to solve every day problems. General Overview*: Week Activity 1 Distribute Biotech in the News Worksheet Due: Biotech in the News Worksheet 2 Class discussion of interesting biotech Groups and Topics Assigned 3 Due: How Does Our Technology Work? Topic List 5 Due: How Does Our Technology Work? Research 6 Due: Biotechnology Controversies Topic List 7 Due: Statement of Legislation 8 Due: Biotechnology Controversies Research 10 Group Presentations Assignments (may vary by instructor): 1. Biotech in the News Worksheet: Graded individually. A worksheet will be distributed during the first lab of the term. 2. Individual Research Assignments (2): Each student will submit a unique assignment. The assignments of members of the same group should not overlap. 3. Presentation: Each group will describe the technology they were assigned. At the end of their presentation, the group will propose some form of regulation for their technology and present two campaign ads; one for and one against this regulation. 4. Team evaluation: At the end of the term each student will anonymously evaluate the participation of each team member. 3

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5 Reference Format for General Biology This class includes a research project and with research comes references. To avoid charges of plagiarism, it is essential that you learn the proper way to cite the source of information in the text of your paper and list complete references at the end of your paper. Plagiarism will result in a score of zero on your assignment. You may already be plagiarizing without knowing it! Get informed! Visit this site for a simple explanation of what is and is not plagiarism: Using References in Science For the most part, scientists do not quote directly from papers but summarize the facts or conclusions of a paper in one or a few sentences that are referenced at the end of a sentence. You must practice summarizing previously published material in your own words. Points will be taken away from your grade for using quotations from rather than summarizing your references. After summarizing the data, cite the publication to which you are referring. In Text Citation format: (Author, Year). Scientific citations include the author and the year of publication but not the page number. Citations are placed in parentheses at the end of a sentence before the period. If one citation covers several sentences, you either place the citation at the end of each sentence or put the citation at the end of your summary (this is the best, most appropriate way to do it). Your Reference List Any resources cited in your text must be listed in proper format in the References section of your paper. Your reference list allows the reader to find more information on the topics you cited in the text of your paper and provides verification for your discussion section. Most scientific publications require a format based on, but not exactly the same as APA (American Psychological Association) format. Examples of format required for this class are provided on the next page. Reference List Format: For the format of different types of references, use the guide on the back of this page. References should be alphabetized by author s last name (notice that all formats list author last name first. Only references actually cited in your paper should be included in the reference list. 5

6 Required Reference Format for General Biology: 1. Journal articles (including those accessed digitally) Author(s). (year) Title of article, Journal name vol: pages. Freas KE, PR Kemp (1983) Some relationships between environmental reliability and seed dormancy in desert annual plants, Journal of Ecology 71: Books Authors(s) (year) Book title, Publisher, City, State, and country where publisher is located. Fenner, M (1985) Seed ecology, Chapman and Hall, New York, NY, USA. 3. Edited books Author(s) (year) Chapter title. In Book Title (names of editor(s), eds.), Publisher, City, State, and Country, pages Kemp PR (1989) Seed banks and vegetation processes in deserts. In Ecology of Soil and Seed Banks (MA Leck, VT Parker, RL Simpson, eds.), Academic Press, New York, NY, USA Websites Author(s)* (year**) Title of Webpage. Retrieved on <date you visited that website> from <complete website URL>. Backyard Gardener (no date) Seed Germination Database. Retrieved on February 8, 2011 from *Find the author at the very bottom of the page. If no author listed, use the sponsor of the website (check the URL for the name before.com/.org/.gov) **Some webpages lack a date of publication. If this is the case, put no date in the parentheses. You should ask yourself if this really is a reliable resource if it doesn t give you a date! Notice that Book and Journal titles are always italicized or underlined. Chapter titles and article titles are in normal font and only the first letter of the first word is capitalized. 6

7 Date due: Name: Pre-lab 1: Cells and Microscopes 1. A. Differentiate between a dissecting microscope and a compound light microscope. (see section 6.1 of your textbook). B. Which type will we be using in today s lab? 2. Explain how you would determine the total magnification of an image viewed with a microscope? 3. Differentiate between a prokaryotic and a eukaryotic cell. Which type of cells are we observing in today s laboratory? 4. Complete the following table regarding the various membrane-bound organelles. You may have to do some Internet investigation to fill in some of the spots! Organelle Name Central vacuole Chloroplast Golgi Body Mitochondria Nucleus Plant, animal or all eukaryotic cells? Function Can you see it with a compound microscope? 7

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9 Name: Lab 1: Microscopes and Cells By the end of this lab you should be able to: Use a compound light microscope Calculate the magnification of an image Prepare a wet mount Describe the basic structure of a cell Identify organelles that are visible with a compound light microscope Exercise 1. Introduction to Microscopes:Most cells are so small that you cannot distinguish between them with only your eyes. The structures within a cell are even smaller. We can explore cells and their internal structure with instruments known as microscopes. Microscopes contain specially shaped pieces of glass (lenses) that alter the path of light as it travels through a specimen to your eye. In this class, you will become familiar with two types of microscopes. A dissecting microscope provides three-dimensional images at lower magnifications. A compound microscope provides two-dimensional details with a wide range of magnifications. Obtain a compound microscope from the cabinet. Always carry a microscope with two hands (one on the base and one on arm). Obtain a diagram of a microscope. As your instructor describes the parts of the microscope and their function, label the diagram and fill in the following chart. Table 1. Microscope Anatomy and Function Microscope part Coarse Focus Adjustment Knob Diaphragm Fine Focus Adjustment Knob Mechanical Stage Adjustment Knobs Objective Function Ocular Stage As you work, label each of these parts in Figure 1 (next page). 9

10 Figure 1. The Binocular Microscope: Label the parts indicated by arrows. Use Table 1 as a guide to the appropriate names of the microscope parts. 10

11 Microscopes are complex instruments that require special care. The following procedures will help you comfortably use a microscope. A. Sitting at your microscope: Microscopes are designed for people who are sitting down with the microscope directly in front of them. Bending over to look in a microscope will hurt your back! 1. Place your microscope directly in front of you. 2. Adjust the height of your chair so that you can easily look into your microscope. B. Getting started with a slide: 1. Plug your microscope in and turn it on 2. Lower the stage completely. 3. Click the low power objective into place. 4. Place the slide into the slide holder on the stage. 5. Use the coarse adjust knob, focus the image you see through the oculars/ 6. Use the fine adjust knob to fine tune the focusing. 7. If necessary, go up in magnification by clicking the medium power objective into place. 8. Use the fine adjust knob to fine tune the focusing. Never use the coarse adjust knob at medium or high power. 9. If necessary, go up in magnification by clicking the high power objective into place. Repeat step 7. C. Getting the best image (critical illumination): In order to get the best view of your specimen (this means actually being able to see organelles within cells!), you will need to learn to set it up appropriately. 1. Use the condenser adjustment knob to raise the condenser to the top (almost touching the stage). 2. Lower the condenser slightly (about ¼ of a turn) 3. Adjust the iris diaphragm (the lever on the front of the condenser) until you see a comfortable level of contrast when looking at a specimen. This level will probably need to be readjusted at every magnification you use. D. Putting away your microscope: 1. Click the lowest power objective lens (4X) in place and use the coarse focus adjustment knob to raise the objective completely. 2. Turn off the light 3. Remove your slide and return it to the proper slide tray. 4. Unplug the microscope and loosely coil the microscope cord in your hand. Drape the coil over the ocular lens(es). 5. Replace the dust cover. 6. Return the microscope to its cabinet such that the arm of the microscope is facing you. Hints for looking at specimens: 1. If you lose your specimen, go back down to low power and start again. It won t take too long and it will save you a great deal of frustration. 2. To avoid losing your specimen, make sure that it is in the center of your field of view before you go up in magnification. 3. If you can t find your specimen, go to the edge of the slide and find the edge of the cover slip. Get the edge of the cover slip into focus. Your specimen should be in the same focal plane as the edge of the cover slip. 4. Do not use the coarse adjust knob at medium and high power. Not only do you risk damage to your slide and your microscope, you are likely to go so far out of focus that you will lose your specimen. 11

12 Exercise 2. Your first slides: 1. Obtain a slide of the letter e. a. Place the slide on to the stage so that it is right side up (so you would be able to read it). b. Examine the slide with the lowest magnification on your microscope. c. Sketch the letter e as you see it through your microscope in the margin of your manual. How does the orientation of the letter e as seen through the oculars of your microscope differ from the actual orientation of the letter e on the stage of the microscope? 2. Examine the letter e with at medium and high powers magnification. What happens to the amount of light in your field of view as you increase magnification of your microscope? What happens to the amount of specimen you can see in one field of view as you increase the magnification of your microscope? 3. Now, compute the total magnification available with each objective of your microscope. a. Record your ocular magnification in appropriate sections of Table 2. b. Record your objective magnifications in the appropriate sections of Table 2. c. Multiply the magnification of the ocular by the magnification of the objective lens. For example, a 10X eyepiece and a 10X lens would provide a total magnification of 100X (10x10). Fill in the following chart (Table 2) for your microscope. Table 2: Total Magnifications for Laboratory Microscopes Ocular Magnification Objective Magnification Total Magnification (Ocular X Objective) 12

13 Exercise 3.Preparing a Wet Mount: The slides most students are familiar with are prepared slides. To make a prepared slide a specimen is preserved, sliced and mounted permanently on the slide for use by many students. Prepared slides usually incorporate stains or dyes that highlight specific parts of the cell or tissue you are examining. In the laboratory you can make your own slides of fresh material by preparing a wet mount. Dyes can also be used to highlight cell structure in wet-mounted specimens. General Procedure: 1. Obtain a clean slide and cover slip. 2. Place a drop of liquid (water or saline solution depending on the type of organism you are examining) in the center of the slide. 3. Place the specimen in the drop of liquid. The specimen should be very small and as thin as possible. 4. Hold the cover slip between your thumb and forefinger. Place one edge of the cover slip on the slide near the left side of the liquid containing your specimen. The cover slip should be at a 45 angle to the slide. 5. Slowly lower your cover slip onto the slide. Prepare a wet mount of a portion of one leaf of Elodea, a pond plant, for examination with your microscope. The small green bodies in the cytoplasm are chloroplasts. Their color is due to the presence of chlorophyll. Move the slide around and find a cell in which the chloroplasts are moving. This is called cytoplasmic streaming. Sketch a small portion of your field of view in the space provided below. a. Indicate the total magnification at which you viewed the slide. b. Label the chloroplast and central vacuole. Elodea Total Magnification: As you examine Elodea with your high power objective, carefully turn the fine focus adjustment knob. What happens? By turning the fine focus adjustment knob, you are looking at different layers of cells within the Elodea leaf. Although we can place a three dimensional specimen under the compound microscope, we can only obtain a two dimensional image of that specimen. The specimens in prepared slides are sliced so thinly that focusing up and down is not necessary. In many living specimens, focusing up and down allows us to examine different slices of a three dimensional specimen. Estimate the number of layers of cells that form an Elodea leaf by focusing up and down on your specimen: 13

14 Exercise 4. Using Stains and Dyes to Examine Cell Structure:In this exercise, you will not observe cells, You will learn how you can use a stain to locate a specific compound found in some cells. Later in the lab, you will refer back to your observations to understand what you are seeing within a cell. Procedure: 1. Place three watch glasses on top of a piece of white paper. 2. Add a very small scoop (less than a pinch!) of sugar to one watch glass. Simple sugars like this are a short-term form of energy storage in cells. 3. Add a very small scoop (less than a pinch!) of cornstarch to a second watch glass.cornstarch contains isolated leucoplasts or starch-storing structures from the cells of corn kernels. Starch is an energy storage molecule produced by plants. 4. Add several drops of iodine solution (IKI) to the empty watch glass. What color is the iodine solution? 5. Add several drops of iodine solution to the watch glass containing sugar. What color is produced by mixing iodine and sugar? 6. Add several drops of iodine solution to the watch glass containing cornstarch. What color is produced by mixing iodine and cornstarch? 7. Create a wet mount using only a few grains ofcornstarch and following the directions in the previous exercise. Observe these leucoplasts with a microscope. Are leucoplasts organelles? Explain your reasoning. 8. While your slide is still on the microscope, stain your sample with iodine solution using the following method. a. Place a drop of the stain on the slide next to the cover slip. b. Twist the corner of a tissue into a point. Place the point at the edge of the other side of the cover slip (opposite the drop of stain). c. Capillary action will draw the stain under the cover slip. What is the iodine actually staining in the cornstarch sample? The fluid or the leucoplast? You might want to sketch what you see in the margins for reference later in the lab. Based on your observations, you should be able to draw a conclusion about how iodine solution can be used in cells. Fill in the blank with your conclusion: Conclusion:Iodine can be used to detect (starch or sugar?) in cells. 14

15 Exercise 5. Plant Cell Structure:Cells are the basic unit of life. Their importance, however, was not understood until scientists were able to actually observe cells using the microscopic techniques above. The rest of this lab is designed to encourage you to explore cell structure with compound microscopes. Although we will be able to see some of the structural components of a cell, many organelles are too small for us to see with these microscopes. Plant cells contain several organelles that are not found in animal cells. All cells are surrounded by a rigid cell wall and contain a central vacuole. Many plant cells contain chloroplasts, a green organelle that is used for photosynthesis. Regions of the plant that do not perform photosynthesis may contain other versions of chloroplasts that perform different functions in the cell. Plant cells also contain a variety of chemicals including the energy storage compounds we discussed in exercise 4. Part of this exercise is to use the information you gained in exercise 4 to determine which energy storage compound (sugar or starch) is present in onion and potato cells. Before you begin, complete the following hypotheses based on what you think the results will be. Onion epidermal cells contain the energy storage compound. Potato cells contain the energy storage compound. A. Onion Epidermal Cells: Go to the onion epidermal cell station in the lab. Carefully follow the instructions at the station to prepare a wet mount of just the epidermis (an outer skin-like tissue) of an onion. Cells exhibit a great diversity in size, shape, and function, but each cell (whether plant or animal) shows a basic similarity in intracellular organization. Perhaps the most obvious structure in the cell is the nucleus. If you cannot see the nuclei, ask your instructor for help. Notice that many of the nuclei appear to be pressed against the cell wall, the outer, nonliving, cellulose envelope enclosing plant cells. The nucleus is restricted to this position because of a large, fluid-filled central vacuole. Draw several onion epidermal cells in the space provided below. a. Indicate the total magnification at which you viewed the slide. b. Label the nucleus and central vacuole of a cell. Usually, plant nuclei are shown pushed to the side of the cell. How would you account for the fact that some of the nuclei in your onion cells appear to be centrally located in the cells? Onion epidermal cells Total Magnification: 15

16 Stain your specimen with Iodine as described in Exercise 4. What happens? Describe your observations in detail! What type of energy storage compound (sugar or starch) is probably present in onion epidermal cells? B.Potato cells.go to the potato station in the lab. Carefully follow the instructions at the station to prepare a wet mount of a thin slice of potato cells. Examine your slide of potato with your microscope. Many cells were cut open by the razor blade releasing their contents. What do you think the oval objects are that are scattered on the slide? Stain the potato cells with iodine using the procedure outlined in Exercise 4. What happens? Describe your observations in detail! Draw a portion of your stained potato in the space provided. a. Indicate the total magnification at which you viewed the slide. b. Label the cell wall and any organelles you see Potato Cells Based on your results, what are the small oval objects within potato cells?hint: Check out the What to Expect explanation at the end of the instructions for this procedure. Total Magnification: 16

17 Exercise 6. Wet mounts of Single-celled Aquatic Organisms:The first organelles evolved in single celled eukaryotes now categorized in to a broad group known as protists. Protists exhibt a variety of organelles; both those that appear in multicellular organism and several unique membrane-bound organelles that are seen in only one or a few species. To examine a few of these unique organelles, let s take a look at one of the larger protistan species, Paramecium caudatum. Procedure: 1. Obtain sample of Paramecium culture with a plastic pipette*. Look at the sample jar. These organisms may be big enough to see with your eyes, but only as tiny moving specks in the culture medium. Observation may show you the best place in the jar to collect a sample. If you don t see any specks, take your sample from near (but not at) the bottom of the jar. 2. Place a drop of your sample on a clean slide. 3. Gently lower a coverslip over the drop as described earlier in the lab. 4. Place your slide on the microscope and focus on the edge of the coverslip. This will be the easiest way to make sure you can find your specimen! 5. Use the scanning objective (4x) to find the organism.paramecium tends to move quickly. Have fun watching them swim for a minute! 6. Slow your Paramecium down. To get a closer look at the organelles in the Paramecium, you will need to both increase your magnification and slow it down. Add a very small drop of methylcellulose (Proto-slo) to the edge of both sides of your coverslip. This substance will slowly diffuse under the coverslip and the Paramecium will slow. If all the Paramecium are gone after adding methylcellulose, you added too much and will need to make another slide. 7. Focus on a Paramecium with the highest power objective.be sure to adjust your contrast with the iris diaphragm so you can see its cilia and organelles! Use the description below to identify the various organelles in this organism. 8. Sketch a Paramecium in the space provided on the next page. Make your drawing BIG! Fill in the space! Label the visible structures highlighted in the description below. * If live Paramecium are not available, your instructor will provide a prepared slide. USE THIS DESCRIPTION TO IDENTIFY CELL STRUCTURES IN PARAMECIUM! One of the first things you may see when you look at this organism is its hairy covering. Paramecium are covered with thousands of tiny cilia. Cilia are extensions of the cytoskeleton surrounded by the plasma membrane. The cilia beat back and forth much like the oars of a boat propelling the cell through its aquatic environment. Paramecium are often called slipper cells because of their shape. As the organism swims, it will roll a bit and you will see a groove slanting downward toward the center of the cell. This structure is the gullet, where food enters the cell. At the base of the gullet, food is captured in tiny vesicles that enter the cell and fuse with lysosomes. Remember that lysosomes (which you cannot identify here) contain digestive enzymes that will break down the food and release the nutrients to the cell. Together, the lysosome and the food vesicle form a larger organelle known as a food vacuole. Food vacuoles may be 17

18 visible in the cell you are examining. The color of this organelle depends on what food the Paramecium is consuming. Inside the organism you will notice several large organelles. The most obvious organelle is the large and usually star-shaped contractile vacuole. The contractile vacuole plays an important role maintaining the structure single-celled fresh water organisms. As we will learn next week, water tends to move into cells by osmosis. If this process were to go unchecked, fresh water organisms like Paramecium would swell and burst! The contractile vacuole found in many protists absorbs water and, when full, dumps it back out into the environment. This is a continuous process and, if you watch long enough, you may be able to see the contractile vacuole fill and dump in the organism you are observing. The other organelle that will be visible in the Paramecium is the large macronucleus. Paramecium actually contain two nuclei. The larger macronucleus is essential for producing the proteins responsible for keeping the cell alive. The smaller micronucleus is essential for reproduction. The kidney-bean shaped macronucleus is usually centrally located in the cell and has a dense or grainy appearance. If a micronucleus is present is will be fairly close to the macronucleus in the indent of the kidney bean shape of the macronucleus. Paramecium caudatum Total Magnification: Describe several ways that Protists like Paramecium differ from the plant cells we have examined so far in this lab. 18

19 Exercise 7: Animal Cell Structure: As you look at a representative animal cell, Compare its structure to that of the plant cells you examined. What is similar? What is different? A. Cheek cells: Go to the cheek cell station in the laboratory. Carefully follow the instructions posted at the station to prepare a wet mount of your cheek cells. Note that instead of mounting your cheek cells in water, you are mounting them in a salt solution. Why do you think that is? Briefly examine your cheek cells with them microscope. Once you have a few cells in focus, leave the slide in position on the microscope stage and stain the cells with methylene blue as described in Exercise 4. Methylene blue is a stain that turns DNA blue. Draw several cheek cells in the space provided. a. Indicate the total magnification at which you viewed the slide. b. Label the nucleus, plasma membrane, and cytoplasm. Did you observevery small blue dots on the surface of your cheek cells. These are bacteria, prokaryotic cells. Note how much smaller they are than the eukaryotic cells we are focusing on in this lab! Human Cheek Cells Total Magnification: B. Cells within organisms:like plants, animals are multicellular and we usually cannot look at isolated animal cells as we did with cheek cells. When arranged as a tissue, animal cells often lack the characteristic boxy shape of the cells we saw in Elodea earlier in the lab and can be harder to identify. The key to finding a single animal cell in a multicellular tissue is to look for the nucleus. As we discovered in cheek cells, the centrally located nucleus of animal cells is pretty large and usually stains darker than the rest of the cell. When you look at an animal tissue specimen, look for these structures and remember that one nucleus = one cell. Note: To understand this material, you must look at the slides in the order described! 19

20 Procedure 1 (liver tissue, standard staining for contrast only): 1. Use your microscope to focus on a sample of liver tissue (slide = Liver section) at the highest power magnification. Remember to center and focus the image with each of the lower power objectives before increasing your magnification! 2. Identify a single cell on your slide. 3. Sketch several cells in the space provided on the next page. Label a single cell and its nucleus. 4. Check with your instructor to make sure you have really found a cell before moving on to the next step. As we learned earlier in the lab, special stains can be used both to increase contrast in a specimen, but also to highlight specific parts or compounds within a cell. To complete our examination of animal cells, we will examine liver tissue, but this time, stained with a special dye that highlights mitochondria, the organelles that produce power (ATP) in the cell. Procedure2 (liver tissue, specially stained for mitochondria): 1. Use your microscope to focus on a sample of liver tissue with stained mitochondria (slide = Mitochondria Amphiuma Liver) at the highest power magnification. Remember to center and focus the image with each of the lower power objectives before increasing your magnification! 2. Identify a single cell on your slide as described above. In addition to finding a nucleus, each cell you see should contain many smaller, darkly stained dots. These are the mitochondria. 3. Sketch several cells in the space provided. Label a single cell, its nucleus and a few mitochondria. Liver Cells Total Mag: Liver Cells with stained Mitochondria Total Mag: 20

21 Applying What You ve Learned: Why is it important to know the total magnification you are using when you are examining a specimen with a microscope? Complete the following table comparing the various organelles observed in this lab. Plant, Protist, Organelle Function Animal or all eukaryotic cells? Nucleus Chloroplast Mitochondria Central vacuole Food Vacuole Contractile Vacuole Compare and contrast the arrangement of cells in multicellular plants (Elodea) and animals (liver) based on what you observed in this lab. Based on what you observed in this lab, how do animal cells differ from plant cells? Describe several roles of stains and dyes in microscopy. Indicate specific exercises in this lab that highlight these roles. 21

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23 Date due: Name: Pre-lab 2: Membranes 1. Differentiate between the processes of diffusion and osmosis. 2. Examine Figure 5.12 in your textbook and read the section on carbohydrates (section 5.7). The glucose we are using in Exercise 2 in today s lab is a monomer. The starch we are using is a polymer. A. Which of these molecules (starch or glucose) do you think is bigger? B. Which of these molecules (starch or glucose) is more likely to travel through a selectively permeable membrane? 3. Use the instructions for Exercise 3 (in this week s lab) to sketch the 4 experimental set-ups your group will create in class. Label the contents of each mock cell. Label the solution that will go on the outside of each mock cell. The first one is done for you. 5% sucrose 25% sucrose Cell A Cell B Cell C A Cell D 4. When a hypertonic solution is separated from a hypotonic solution by a selectively permeable membrane, which direction does water move? Water moves from the solution to the solution. 23

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25 Name: Lab 2: Diffusion and Membrane Transport Diffusion is a process in which molecules and particles move by random collisions and eventually become randomly distributed throughout a solution. The result of this is the apparent movement of those particles from high concentration toward low concentrations of the substance that is diffusing. We say that the particles are diffusing along a concentration gradient from high toward low concentration. By the end of this lab, you should be able to: Describe how diffusion takes place Explain the similarities between diffusion and osmosis Explain how size relates to the ability of materials to cross membranes Describe the relationship between cell shape, size and function Exercise 1. Cycles of Life Program 3(Permeable Packaging): Fill in the blanks as you watch this short film. 1. Between the... inner cell and the... outside world stands the cell s. 2. The lipid molecules of the membrane naturally assemble in a layerbecause their tails water as their heads attract it. 3. Nature composes the (cell) membrane with a combination or of different lipids, carbohydrates and proteins. These molecules are not stationary, they constantly move changing their position. 4. Diffusion is a fundamental physical process. Smaller molecules move directly through the membrane as they travel from a concentration to a concentration. This process occurs naturally regardless of life. 5. Some proteins in a cell s membranes act as that allow specific large and/or charged molecules to cross passively. 6. In passive diffusion passive transport the cell requires no. 7. When cells transport substances from a lower concentration to a higher one, they use proteins that act like a pump and need a boost from an molecule. 8. In endocytosis, part of the surface membrane surface material, forming a (vesicle) which brings contents the cell. 9. In exocytosis, the sac forms internally, travels through the cytoplasm to the membrane and fuses with the membrane releasing the contents the cell. 25

26 Exercise 2. Brownian Motion: All molecules are subject to naturally occurring movement. Under the microscope, this movement will appear like a gentle vibration. 1. Put a drop of carmine dye suspension on a slide. Add a cover slip and place the slide on a microscope. Let it sit for a couple of minutes to settle and for the cover slip to level out. 2. Observe the smallest particles on high power. If you see all the particles on your slide moving across your field of view in the same direction, you should wait a bit longer. The movement you are seeing is the flow of water across the slide. Ignore this movement! When the slide has settled, you will be able to observe the movement of individual particles. What are the particles doing? Look at a single particle. Does it always move in a single direction? Describe the path it seems to follow. This movement was first observed by Robert Brown who concluded that the particles must be moving because water molecules were colliding with them (in spite of the fact that you cannot see a water molecule). Exercise 3: Diffusion: Membranes form the boundary of the cell and the boundary for most cell organelles. They regulate the movement of materials between a cell and its environment and between chambers within the cell itself. Do all molecules pass readily through a membrane? What determines which particles will pass through a membrane and which won t? Procedure: 1. Fill a 150 ml beaker with water to within 2 cm of the top. Add enough IKI (iodine) solution to turn the beaker contents a medium golden-brown. Put about 1 cm of this solution in a test tube for use in the controls section (below). 2. Obtain a 15 cm piece of soaked dialysis tubing. Tie one end of the tube into a knot (the knot should be as close to the end of the tubing as possible). Roll the tubing between your fingers to open it up to a bag. 3. Use a dropper to pour starch solution into the bag to a depth of about 2 cm. 4. To the same bag add an equal amount of glucose solution. 5. Rinse off the outside of the bag to remove any spills. 6. Hang the dialysis tubing over the side of the beaker with the bag hanging in the iodine water. Hang the other end outside of the beaker and use a rubber band to hold it in place. 26

27 7. Set your beaker aside for about 30 minutes. While you wait, prepare the Control test tubes. Controls: As you have probably learned in lecture, scientists use experiments, like the one you re doing now, to test their hypotheses. In an experiment, the conditions that a sample is exposed to are changed slightly and observations are made as to how the sample responded to that change. Experiments are both controlled and contain controls. Controlling an experiment means that you maintain constant conditions (temperature, amount of solution, level of light, etc) for all experimental treatments and controls. Generally, only one condition is changed at a time (the experimental treatment). A control is a treatment in which the condition being tested by the experiment is held constant. By including controls in their experiments, scientists can compare the results of experimental treatments to their standard to determine if their treatment actually causes a change. Remember that the control is separate from the experimental sample. Conditions change in an experimental treatment, but are maintained in a control. That being said, controls are often classified as being positive or negative. A positive control is a one that is expected to show a definite change. For example, the dye methylene blue stains DNA blue. A positive control for an experiment testing for the presence of DNA might be a mixing purified DNA with Methylene blue so that you can observe the exact color. Negative controls are expected to show little (or slow) change. In the case of Methylene blue and DNA, we might combine Methylene blue with a purified protein as a negative control to show that only DNA stains with this dye. Together, the positive and negative controls can be compared to each of the experimental treatments to determine if an experimental treatment had any affect. Return to lab 1, exercise 4 to answer the following questions. What was the positive control for the experiment performed in lab 1, exercise 4? What was the negative control for the experiment performed in lab 1, exercise 4? Prepare the controls for today s experiment Prepare 4 the four test tubes described in Table 1 by adding 1 cm of the required solutions to each tube. See the section on Benedict s reagent below for the proper procedure. Before continuing on to the Interpreting Results section, complete the table by explaining why each of these controls is important to this experiment. 27

28 Table 1: Controls for Diffusion Experiment Tube # 1 Starting with IKI + water (your beaker solution) Add equal amount of Starch stock solution Result of Reaction Purpose of this control 2 Glucose stock solution IKI 3 Glucose stock solution 4 Starch stock solution Benedict s Reagent & Heat Benedict s Reagent & Heat Benedict s reagent Procedure:Benedict s reagent is a chemical commonly used in medical labs to indicate the presence of glucose in bodily fluids like urine. The presence of high levels of glucose in urine is a symptom of diabetes. We will use Benedict s reagent to test for the diffusion of glucose. Use the following procedure: 1. Fill a 400 ml beaker with 2-3 cm of water. Place the beaker on a hot plate and bring the water to boiling. 2. Mix an equal volume of the solution to be tested and Benedict s reagent in a test tube 3. Place the test tube in the boiling water for 5 minutes. If glucose is present, you will see a color change. Analysis (Do not clean up yet! You have one more experiment to go!): After letting the experiment sit for 30 minutes, examine the contents of the bag. Can you tell if starch has moved out of the bag? Describe any observations that helped you answer this question. Can you tell if iodine has moved into the bag? Describe any observations that helped you answer this question. Explain how the control test tubes helped you understand these results. 28

29 You also need to determine if glucose moved out of the bag. Write out the step-by-step procedure your group will use to determine if glucose moved out of the bag. Hint: look at the supplies provided in your kit for this lab! Perform the procedure you described above. Has glucose moved out of the bag? Describe any observations that helped you answer this question. In relative terms, a molecule of iodine is small, one of glucose is medium sized and a molecule of starch is very big. What can we infer about the structure of the dialysis membrane we used in this experiment based on the results of our experiment? Exercise 4. Osmosis: Water molecules move along concentration gradients just like solute molecules dissolved in the water, but they move in opposite directions. Water moves from regions of low solute ( high water ) concentration to regions of high solute ( low water ) concentration. The movement of water across membranes known as osmosis. Procedure (Modified from Glick et al., The Process of Science: Seven Studies of Life): 1. Soak your dialysis tubing in distilled water for a minute or more. You will be using 4 pieces of tubing to make 4 mock cells. Tie the tubing in a knot about 2 cm from one end. Open the other end of the tubing by rubbing it together between your fingers. 29

30 2. Write the letters A, B, C, or D in PENCIL on four tiny pieces of paper (they must fit into your dialysis tubing). 3. Fill the bags about half full with the following solutions. After filling the bag, place the appropriate label inside: A. 5% sucrose B. Distilled water C. 10% sucrose D. 25% sucrose 4. Force as much air out of the bag as possible by placing it between two fingers and moving your hand away from the solution in the tubing. Tie the open end of the tube in a knot. The bag should be limp after it is tied. Based on what you ve learned about osmosis, why would it be a bad idea to tie your bag just above the solution or filled with air so that the bag is turgid? Hint: Read the introduction to exercise 4! 5. Squeeze your bag gently to check for leaks. If you find a leak, re-tie your bag. 6. Rinse your bag with tap water and blot it on a paper towel to remove excess water. 7. Place a weigh boat on a digital scale and press the zero or Tare button. This will set the weight of the scale (and the weigh boat) to zero. Make sure the scale is set to measure grams (g). 8. Place your cell in the weigh boat and record its weight to the nearest 0.1 g in Table 2 (below) in the column labeled Weight at beginning of experiment. Cell Table 2: Weight change over time during osmosis experiment Predicted weight change (+,, 0) Weight at beginning of experiment (grams) Weight at end of experiment (grams) Net change in weight (+,, 0) A B C D 9. When all the bags for your group are ready, place each bag into a 200 ml beaker of solution as follows. Your beaker should contain about 80 ml of solution (just enough to cover your bag). A. Put bag A into 25% sucrose B. Put bag B into distilled water C. Put bag C into distilled water D. Put bag D into distilled water Put all of your bags into their solutions at the same time! 10. Once your experiment is running, make a prediction about how the weight of each bag will change over the course of the experiment in the column labeled Predicted weight change in Table 2. You can put a + for weight will increase, a -" for weight will decrease" and a 0 for "weight will not change." You do not need to predict the amount of weight a bag might gain or lose. 30

31 11. After 1 hour remove the bags from their solution and blot excess water away from the bag with a paper towel. Weigh the bag to the nearest 0.1 g and record your results in Table 2 in the column labeled Weight at end of experiment. Indicate weight gain for each bag with a +, weight loss with a - and no change in weight with a 0 in the column labeled Observed weight gain. 12. Place the 25% sucrose solution from Beaker A into the Recycled 25% Sucrose container. All other solutions can go down the drain. Your mock cells can be placed in the garbage. Analysis: Is any of the samples in this experiment a negative control. Explain your reasoning. Is any of the samples in this experiment a positive control. Explain your reasoning. What caused the change in weight of your mock cells in this experiment? Is the change the result of the movement of sugars or the movement of water? Explain your answer. Explain the weight change in each bag based on the principles of osmosis. If the weight of a bag did not change, explain why. In your explanation, use the terms hypertonic, hypotonic and isotonic. Bag A: Bag B: Bag C: Bag D: 31

32 Exercise 5. Osmosis in Elodea: The plant cell wall and the central vacuole play an important roll in regulating osmosis in plants. Water that enters a plant cell through osmosis is stored in the central vacuole. As the central vacuole swells with water, it pushes against the cytoplasm and the cell wall (this is why all the chloroplasts are located at the outer edges of a plant cell). Since the cell wall prevents plant cells from expanding, the influx of water creates pressure and pressure inhibits osmosis. The pressure that builds up in plant cells is known as turgor pressure. Turgor pressure is the force that keeps plants rigid. When a plant does not have enough water, it cannot maintain pressure causing it to wilt. We can actually see the response of plant cells to osmosis by subjecting the cells of Elodea to different solutions. Your instructor has set up two microscopes with samples of Elodea. In one, the Elodea is bathed in pure water. In the other, the Elodea is bathed in a highly concentrated salt solution. 1. Which microscope (your instructor will have them labeled microscope A and B) contains Elodea bathed in a high salt solution? 2. Explain your reasoning using the terminology appropriate to discussions of osmosis. 3. If you were to immerse an entire Elodea plant in salt water instead of just a few cells, how would the plant change? Hint: consider the role of pressure in maintaining the structure of a plant 32

33 Applying What You ve Learned: When evaluating the validity of an experiment, scientists always consider the control. The results of an experiment that lacks control or has poorly designed controls are often questioned or rejected. Why are controls so important? Most non-scientists get their information about current research from blurbs on the news. These blurbs seldom consider or discuss controls. Should you accept these news stories as facts? Explain your reasoning. How is Brownian motion (Exercise 2) important to the understanding of diffusion and osmosis? What factors govern the diffusion of a molecule across a membrane? We learned that osmosis is the movement of water across a membrane. Besides a basic need for water, why is the rate of osmosis important to a cell? 33

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35 Date due: Name: Pre-lab 3: Mitosis and the Cell Cycle 1. Interphase Label the following stages of the cell cycle on the pie chart to the left: G 1 G 2 S Anaphase Metaphase Telophase Prophase Cytokinesis Mitosis 2. Complete the following chart comparing the occurrences in the different stages of mitosis. Some boxes have been completed for you. Phase Nucleus Chromosomes Spindle Fibers Prophase The spindle apparatus begins to form. Spindle fibers attach to the centromeres of duplicated chromosomes Metaphase Anaphase Telophase Sister chromatids reach opposite spindle poles and begin to decondense Turn over for one more question! 35

36 3. Carefully read Exercise 4 in this week s lab. Briefly describe how we can estimate the length of time a growing onion root tip cell spends in each of the phases of the cell cycle. 36

37 Name: Lab 3: The Cell Cycle & Mitosis By the end of this lab, you should be able to: Describe what happens during each phase of the cell cycle Use chromosome models or drawings to explain the process of mitosis to another student Identify the stage of the cell cycle of a dividing cell Explain the differences between cell division in plants and animals Exercise 1. Modeling the Cell Cycle with Play-Doh : The following exercises take you through the steps of the cell cycle using chromosomes made of Play-Doh as a model. Chromosome Anatomy: Most cells in your body are diploid; they contain two copies of every chromosome. One copy of each chromosome comes from your mother and the other copy comes from your father. Chromosomes of the same type have the same shape and centromere position (and have the same genes). Chromosomes of different types have different shapes and centromere positions. Different types of chromosomes carry different genes. You will model the cell cycle in a cell with a total of 4 chromosomes; two copies of each of two types. To differentiate between the two types of chromosomes, you should make them different lengths. 1. Obtain 2 jars of Play-Doh.The jars should be different colors. 2. Divide the contents of each jar in half. Put one half of each color back in its jar for now. 3. Use the Play-Doh remaining on the counter to make one long snake and one short snake of each color. You should now have 4 total snakes, two long (one of each color) and two short (one of each color). 4. Begin by looking at one snake. This snake represents a chromosome, a piece of DNA containing many genes. 5. Create four small balls of Play-Doh (about the width of your snake) of a different color (a third color, borrow a bit from a neighbor!) and place the ball in the center of each chromosome. This structure is the centromere. The centromere is a complex of proteins that binds microtubules and is important in the physical separation of sister chromatids (read on to find out about these). Are the chromosomes you built duplicated or unduplicated? The cell cycle 1. Begin with a cell in G 1 of interphase. Use a piece of chalk to draw a big circle on your desktop (this represents a cell).draw a circle inside this circle to represent the nucleus. The circles should be big enough to fit your chromosomes inside! 37