General Biology Lab Manual - F2016 PDF [PDF]

Zitiervorschau

GENERAL BIOLOGY LABORATORY MANUAL FOR LIFE 1010 FALL 2016 UNIVERSITY OF WYOMING TABLE OF CONTENTS LAB 1: INTRODUCTION TO LAB

P.1

LAB 2: SCIENTIFIC INQUIRY

P.3

LAB 3: MEMBRANES

P.11

LAB 4: MICROSCOPES & CELLS

P.19

LAB 5: MACROMOLECULES & ENZYMES

P.31

LAB 6: FERMENTATION

P.39

LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

P.53

LAB 8: DNA

P.61

LAB 9: MITOSIS & MEIOSIS

P.69

LAB 10: GENETICS

P.75

LAB 11: EVOLUTION & NATURAL SELECTION

P.87

APPENDICES LAB NOTEBOOK GUIDELINES

P.97

LAB REPORT GUIDELINES

P.101

EXAMPLE LAB REPORT

P.105

LAB REPORT RUBRIC

P.111

GENERAL BIOLOGY LAB MANUAL – LAB 1: INTRODUCTION TO LAB

LAB 1: INTRODUCTION TO LAB During the first lab period, you will meet your lab instructor and classmates. You will also go over the lab syllabus and schedule, and take an assessment survey. Before the next lab, make sure you have a bound (non-spiral) laboratory notebook, and read the syllabus and pre-lab reading for LAB 2: SCIENTIFIC INQUIRY. Both documents will be covered on your first lab quiz.

1

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY

LAB 2: SCIENTIFIC INQUIRY Learning Objectives 1. Develop alternative and null hypotheses that are testable and correctly address the dependent and independent variables of an experimental question. 2. Design an experiment with a control group and replication to test hypotheses about a factor that might affect plant growth. 3. At the end of a multi-week experiment, demonstrate an understanding of the experiment and the scientific method by preparing an individual lab report with primary literature used for support. 4. Practice unit conversions.

Pre-lab Reading

From Textbook (Freeman et al. 2014): Section 1.5 Science as a Process: The Purpose of Lab In general biology laboratory you will get a chance to explore concepts introduced in lecture in more detail, and get hands on training with scientific tools and procedures. You will also engage in scientific inquiry by developing hypotheses and experiments, collecting data, and arriving at conclusions based on this data. The goal of scientific inquiry, or investigation, is to better understand the world around us. By engaging in inquiry, you are doing science. At its root, science is a process that uses observation and reason to investigate and explore the physical world. This contrasts with the popular definition that considers science to be the organized body of knowledge about nature and the universe – that is, a collections of facts. In reality, science encompasses both definitions – it is both knowledge and process. In lab we will emphasize the ways in which we make observations and use reason to produce knowledge (i.e. the process of science). But to do so requires an understanding of what has already been discovered, so we will also explore the foundations of biology, and reinforce what you learn in lecture. Science proceeds by building upon previous findings, which are published in peer-reviewed journal articles, called primary literature because they present original ideas and new findings (textbooks and other material that summarize and synthesize primary literature are considered secondary literature). Peer-reviewed journal articles can be difficult to read, and becoming proficient at doing so takes a good deal of practice. As a science major, you will be expected to use peer-reviewed literature to support assertions you make in written work, including the lab report you will write for this lab. We will show you how to find peer-reviewed articles and discuss how to read these articles. Any good scientific research project begins with a question – why or how does a particular phenomenon occur? Questions may arise from observations, literature, or discussion with other scientists. Once a question has been selected, we develop hypotheses, which propose potential answers to our question. Hypotheses are educated guesses, based on what we already know, and a good scientific hypothesis is both specific and testable. Hypotheses are the starting point for further investigation, and it is important that they are testable so that they can be evaluated by experimentation and careful observation. Once hypotheses have been formed, experimental or observational studies are designed to test the hypotheses. Well-designed studies attempt to keep conditions as constant as possible, include control groups, and use replication. Experiments or observations are then conducted, and data is carefully recorded. 3

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY Frequently experiments and studies do not proceed as we expect the first time, and must be redone. For example, we might find that the instrument or technique we planned to use does not work as expected. Much science is carried out by trial and error. After all, if we knew what we were doing, it wouldn’t be research! When data have been successfully collected, they are used to evaluate hypotheses. Scientists usually analyze data with statistical tests, which help us decide if our hypotheses are supported or not. Statistical tests can reveal, for example, whether observed differences we see between control groups and experimental groups are likely to be real or merely the result of coincidence. Statistical tests are scientific tools as important as microscopes, spectrophotometers, and pipettes. This semester, to introduce you to statistical tests and their usefulness, we will show you how to perform and evaluate one of the most common and simple statistical test used by scientists, the t-test. In future science classes you will learn about other tests used for different types of data, and you may be required to take a statistics class for your major. After hypotheses have been evaluated and data has been interpreted, we must communicate findings. Remember, science builds on previous work – to advance our knowledge we must share our findings with others so they can build on our work. Scientific findings are shared through peerreviewed journal articles. In fact, the production of articles is one of the main ways in which the success of a scientist is measured. Your professors and teaching assistants, as professional scientists, are expected to publish in scientific journals. The lab report you will write in this class mimics a scientific journal article, and will help you learn to read and write scientifically. We will assemble a lab report in pieces and provide you with constructive feedback at each step. Variables, Hypotheses, and Predictions When we design an experiment or observational study, we are interested in measuring the effect of an independent variable (sometimes a set of independent variables) on a dependent variable. The independent variable is a variable that is changed or manipulated in an experiment, and different levels of an independent variable are called treatments. The dependent variable, also called the response variable, is the variable that is measured. One way to remember the difference is that, hypothetically, the value of the dependent variable depends on the value of the independent variable. For example, consider an experiment designed to address the question: How do omega-3 fatty acid supplements effect blood cholesterol levels? In this experiment a researcher would measure the blood cholesterol levels of the subjects, give the subjects an omega-3 fatty acid supplement each day over a two-week period, and then measure blood cholesterol levels of the subjects again. The independent variable would be the amount of omega-3 fatty acids given, and treatments would be the different doses of omega-3 fatty acids, such as 50 mg/day, 100 mg/day, and 200 mg/day. The dependent variable would be the change in the blood cholesterol levels of the subjects over the twoweek period. Hypotheses are potential answers to scientific questions. In the context of an experiment, they suggest possible outcomes. This semester we will develop an alternative hypothesis and a null hypothesis for each experiment we perform. The use of the two types of hypotheses may seem a bit odd at first, but they are used because they facilitate the use of statistical tests. The alternative hypothesis (sometimes called the research hypothesis) states that the independent variable has an effect on the dependent variable. For the example above, the alternative hypothesis (often noted as Ha) would be that the amount of omega-3 fatty acids taken has an effect on blood cholesterol levels. The null hypothesis states that the independent variable has no effect on the dependent variable. For the example above, the null hypothesis (often noted as Ho) would be that the amount of omega3 fatty acids taken has no effect on blood cholesterol levels. 4

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY Note that the alternative hypothesis says nothing about the nature of the effect of the independent variable on the dependent variable. For example, taking more omega-3 fatty acids might decrease blood cholesterol levels or increase blood cholesterol levels (or might have no effect on blood cholesterol levels). We can make a prediction to state what we results we expect to see based on what we already know. For example, if it has previously been found that diets high in omega-3 fatty acids, such as those rich in fish and nuts, are correlated with high levels of cholesterol, we might predict that taking more omega-3 fatty acids will increase blood cholesterol levels. Pre-lab Questions Each week, you will begin lab by taking a group quiz and answering starting questions with your classmates. You should read and consider the questions below before lab so you are better prepared. 1. Can you outline the scientific process as presented above? 2. If given an experimental question, could you identify the dependent and independent variables? Could you identify treatments? 3. If given an experimental question, could you develop alternative and null hypotheses? 4. If given an experimental question, could you make a prediction? 5. When designing an experiment, what are some important considerations? Come up with some general guidelines for experimental design.

5

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY

Exercise A: Begin Plant Growth Experiment During the next five weeks you will design and carry out an experiment that will form the basis for your lab report and other assignments throughout the semester. Question: How does the addition of nutrients affect plant growth? Your goal is to investigate how some factor or treatment affects plant growth. Plant growth can be measured in a variety of ways. For this experiment we will measure plant height and count leaves each week, and measure plant mass at the end of the experiment. You may work in groups of 4 or more, and each student may plant two pots. Starting Questions Discuss the following questions with your group and record your responses in you lab notebook. 1. What are some factors you think might affect plant growth? Come up with at least three factors. 2. Pick one of the factors you came up with that might affect plant growth. In an experiment to test the effect of this factor on plant growth, what is your independent variable? How about your dependent variable? 3. Write an alternative hypothesis and a null hypothesis for an experiment that tests the effect of your chosen factor on plant growth. Available Materials Light banks Pots and trays Bins (2 per section, control & treatment) Seeds for various plants Sand Potting soil Sieves to rinse sand to remove salts Measuring cups Perlite Vermiculite

Coffee filters (4 cup basket style) Osmocote ® slow release fertilizer Nitrogen/Phosphorous/Potassium Scales Weighboats Spatulas/forceps Plant tags Sharpies Large beakers for watering

Experimental Design After you have decided on an experiment to perform, record the following in your lab notebook: Objective, Variables, Hypotheses, Prediction, Control and Treatment groups, and Procedure. Refer to the lab notebook guidelines (pages 97 – 99).

6

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY Basic Planting Procedure You may choose to alter this basic procedure for your group experiment. If so, be consistent and record any alterations you made. Planting (Fig. 1): 1. Line a pot with two coffee filters. The coffee filters hold sand in the pot. 2. Rinse 250 mL of sand per pot for 1 minute. This is done to remove salts that might inhibit plant growth. What size sieve did you use to rinse sand? 3. Fill the pot half-way with sand (~125 mL). 4. Add 2.5 g of Osmocote® slow release fertilizer. If you are preparing a pot for a treatment group in which you are manipulating nutrients, add the extra nutrients now too. How much extra nutrient did you add to treatment pots? 5. Fill the pot the rest of the way (to the bottom of pot rim) with sand. 6. Make a small divot (~0.5 cm deep) with your thumb. Place two seeds in the divot. 7. Cover the seeds and pot surface with a thin layer of vermiculite 8. Place a plant label in your pot. See Fig. 2 for labeling instructions. 9. Place pot a tray for your lab section under a growth light. How many pots did you prepare for each group?

Fig. 1. Diagrammatic planting methodology for Plant Growth experiment. Use coffee filters in place of fabric. Nutrient addition is only required if preparing a pot for the treatment group.

7

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY

Fig. 2. Example of a plant label. Your plant label should include your lab section (circled please), treatment (phosphorous addition in example), and your last name. Table 1. Schedule for Plant Growth Experiment and Lab Report Preparation. Week 12 – 16 Sep 19 – 23 Sep 26 – 30 Sep 3 – 7 Oct 10 – 14 Oct 17 – 21 Oct

Plant Growth Experiment - Design experiment - Plant seeds - Thin pots if necessary - Take plant measurements if possible - Thin pots if necessary - Take plant measurements - Take plant measurements - Take plant measurements - Harvest and dry biomass - Weigh dried biomass

Lab Report Preparation HW 1: Methods

HW 3: Scientific Literature HW 4: Introduction HW 5: Data Analysis & Results

31 Oct – 4 Nov

HW 6: Discussion

14 – 18 Nov

HW 8: Peer Review

28 Nov – 2 Dec

Lab Report due

8

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY

Exercise B: Unit Conversions Quantitative skills are essential for biologists. Statistics and mathematical models are important tools scientists use to describe and understand biological systems. To develop your ability to understand and use such approaches, we do calculations and other quantitative exercises in lab. Conversion of units are everyday tasks for many individuals in biological fields. For example, a nurse or doctor may need to calculate the volume of medicine a patient should receive based on the concentration of that medicine. Failure to make such a conversion correctly could have dire consequences. Unit conversions are used to convert the some quantity from one unit of measurement to another. To avoid errors during conversions it is best to use the method demonstrated in the following example. This method allows you to carefully cancel units out so that you are left with your starting quantity (2 days in this example) expressed in the units you want (seconds in this example). For example, convert 2 days to seconds: 1. Write your starting quantity, then multiply it by conversion factors to arrive at your desired unit. Conversion factor can be inverted (

; with this in mind, set-up

conversions so that units cancel out:

2. Once you’ve cancelled units you should be left with only the desired unit(s), in this case seconds:

3. Now simply multiply the numbers:

Conversions factors work because you are simply multiplying your starting quantity by one, without actually changing the quantity. For example, the conversion factor is equivalent to 1: 



9

GENERAL BIOLOGY LAB MANUAL – LAB 2: SCIENTIFIC INQUIRY With your group, perform the following unit conversions. Record your work in your lab notebook. You must show all of your work to receive full credit. 1. 2. 3. 4. 5. 6.

Convert 0.12 m into mm Convert 800 μL to mL Convert 3.5 in to cm Convert 0.5 m2 to cm2 Convert 2.0 miles per hour to m/s Convert 0.2 g/mL to g/L

Clean-up Before you leave lab:  Make sure your plants are well-labeled and placed in the tray for your lab section.  Return any items at your table to their place on the lab benches.  Wipe down tables and benches as necessary to remove sand and soil.

Homework Assignment 1: Methods This week you will prepare a methods section based on the experiment you setup today and submit it to WyoCourses. Refer to the lab report guidelines, example lab report, and lab report rubric (pages 101 – 112) as you prepare your methods section and other homework assignments. This methods section, with some modifications and additions, will be incorporated into you final lab report.

Acknowledgments This lab was designed by Trace Martyn and Chris North. Tim Aston and other graduate teaching assistants have provided expertise and suggestions.

10

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES

LAB 3: MEMBRANES Learning Objectives 1. Compare and contrast the processes of diffusion and osmosis. 2. Design an experiment to test the effects of solute concentration and other variables on osmosis across a model membrane. 3. Understand and apply the terms for osmotic concentration. 4. Consider the utility of models in science.

Pre-lab Reading

From Textbook (Freeman et al. 2014): Sections 6.1 – 6.3 The movement of small and large molecules into and out of cells and their organelles is regulated by selectively permeable membranes. These membranes are an essential part of a cell’s structure, allowing the cell to maintain homeostasis within its environment. The Selectively Permeable Membrane Every cell is surrounded by a thin boundary layer that separates the cell from its environment and maintains its individuality. However, to stay alive, the cell must communicate with its surroundings by allowing energy, matter and information to pass through the boundary layer. To maintain homeostasis, the cell requires the intake of nutrients and gases from the environment, and the export of cellular waste products and compounds destined for other cells. A selectively permeable membrane facilitates these activities. The unique structure of the cell membrane is directly related to its function. The current model for a biological membrane is the fluid-mosaic model. This model describes a fluid state phospholipid bilayer, interspersed with proteins and carbohydrates. The hydrophobic ends of the phospholipids face the interior of the membrane, and the proteins and hydrophilic ends of the phospholipids are free to interact with the intra- and extra-cellular environments. The biological membrane’s specific molecular architecture results in selective permeability to different molecules; some substances cross the membrane more easily than others. Not all molecules pass through at the same rate, depending on size, charge and chemical composition. Movement of molecules In any system, molecules tend toward a state of entropy, or disorder. In a biological system, molecules tend to move toward a state of equilibrium as entropy increases. For example, if a drop of perfume is released into a room, the molecules of the perfume will tend to move outward from the original drop until they are equally dispersed about the room. The molecules have moved from a state of relative order (all in one small drop) to disorder (randomly scattered throughout the room). Thus, molecules tend to move spontaneously down a concentration gradient, from higher to lower concentrations (towards an equal distribution). This spontaneous movement of molecules is called diffusion. The energy for this process comes from the intrinsic kinetic energy (also called thermal motion or heat) of the molecules. We often categorize movement of molecules across the cell membrane into two basic categories – those types of movement which require cellular energy to allow movement from one side of the membrane to another, and those processes which require no cellular energy. 11

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES Those forms of movement requiring energy are known as active transport and must be used to move molecules up (or against) a concentration gradient across a membrane. In active transport, the molecules to be transported across the membrane combine specifically with a transporting structure. This interaction must also be coupled to an energy-yielding chemical reaction or flow. As with other processes which require cellular energy, ATP (adenosine triphosphate) supplies the energy for most active transport. ATP releases free energy when its high energy phosphate bonds are hydrolyzed. It is an essential “common currency” form of energy that an organism can use to drive cellular reactions. Diffusion Two forms of passive transport are diffusion (also known as simple diffusion) and facilitated diffusion. Passive transport requires no ATP, but does require that molecules move down a concentration gradient, from higher to lower concentration as they cross the membrane. Molecules move through the membrane structure in diffusion by random molecular motion after dissolving in the membrane material or moving through small pores or channels. As its name suggests, facilitated diffusion requires that molecules combine with specific transporter proteins (“facilitating,” or helping the movement of molecules) which provide a pathway through the membrane for the molecules. Osmosis Water is considered a nearly universal solvent in biological systems and plays a critical role in biological processes. A special case of passive transport is the diffusion of water across a selectively permeable membrane, known as osmosis. Like other substances, water tends to move down a gradient from a higher concentration of water to a lower concentration of water. Osmotic Concentration The osmotic concentration of a solution refers to its solute concentration, or the concentration of substances dissolved in a solution. It does not refer directly to the quantity of water molecules. Rather, it is an indirect indication of a higher or lower concentration of water molecules; in any solution, as the concentration of solute increases, the concentration of water molecules in that solution will decrease. The terms for osmotic concentrations are relative, that is, they only have meaning in a comparative sense: hypotonic, isotonic, and hypertonic. (Hyper- and hypo- mean “more” or “less” respectively, and iso- means “same.”) A hypotonic solution has a lower solute concentration in comparison to another solution; the hypertonic solution has a higher solute concentration than the solution to which it is compared. Isotonic solutions have equal concentrations of solutes, though the composition of solutes need not be the same in both solutions. A cell in your body is isotonic to the solutions surrounding it, even though the ions in the cell and in solution may be different. Tap water is ____________________to distilled water. Distilled water is ____________________to sea water. Tap water is ____________________to sea water.

12

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES Scientific Models Scientific models are representations of features in the natural world, just as a model airplane is a representation of an actual airplane. They are used to better understand, study, or simulate the feature they represent. One conceptual model already mentioned is the fluid mosaic model, which describes the cell membrane in a way that allows us to better conceptualize a feature of living organism that is difficult to see. Models may also be quantitative and use mathematical equations to represent natural processes. For example, the exponential growth equation (Nt = N0ert) is a model that represents how populations of organisms, even people, may multiply at an increasing rate. Model organisms are well-characterized organisms amenable to experimental manipulation that are studied to provide insights on other species. For example, mice are often used to represent humans in drug trials. Brassica rapa, which you are familiar with as broccoli, kale, cabbage and other vegetables, is a model organism that can represent plants in general. Another well-known model organism is the fruit fly. Many model organisms grow and reproduce quickly. A model may also be a physical representation of an object that shares some features with another object. In today’s lab you will be using bags made of dialysis tubing to represent the cell membrane. Usually made of cellulose, and semi-permeable like cell membranes, dialysis tubing is used in a variety of settings to separate molecules by size. Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. 1. Can you compare and contrast passive and active transport? 2. How is osmosis different from diffusion? 3. If two solutions were described to you, could your compare them in terms of osmotic concentration? 4. What is the main type of macromolecules are the main component of cellular membranes? 5. Can you describe the fluid mosaic model? 6. What factors are likely to affect the rate of diffusion of a substance?

13

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES

Exercise A: Osmosis Across a Selectively Permeable Membrane Osmosis of water across a selectively permeable membrane will depend on the concentration of solutes and water on either side of the membrane. Remember that water follows its own concentration gradient, which is opposite of that of the solute concentration gradient. In this exercise you will generate a hypothesis relating to the movement of a solute (sugar) across a model membrane, and then run an experiment to test that hypothesis. Your instructor will describe the materials available to your group. Write a hypothesis about osmosis across your model membrane (dialysis bag). Then design and run an experiment to test your hypothesis and analyze your results. Assume that the sugar in the sucrose solutions will not cross the membrane.) Available Materials Dialysis tubing P-5000 micropipettes and tips Unwaxed floss Weigh boats Scissors

20, 40, and 60% sucrose colutions Digital scales 6 – 250 mL beakers per group 1 – 400 mL beaker per group Water bath and ice, for heating and cooling

Methodology 1. Soften the dialysis tubing by soaking the pieces in a 400 mL beaker of distilled water for about 5 minutes 2. Fold one end over about 1/2” and cinch with a piece of floss. Tie a square knot tightly, so that the bag will not leak (see Fig. 1). This will be the bottom of each bag. 3. Number the six 250 mL beakers: #1, #2, #3, #4, #5, #6 with lab tape and a sharpie. Do not forget to include at least one control. Also place a small numbered tape “flag” on the top tie of each bag which corresponds to the beaker (sample) number. 4. Carefully pipet 5 mL of solution into each dialysis bag, filling each bag approximately 1/2 full. Fold over the top of each bag, and tie the bag shut tightly with floss. The bags should be limp and filled with about equal amounts of liquid. 5. Record the initial mass of the bag on the balance. Record the initial masses in a wellorganized table with space to record masses for each bag every 15 minutes 6. Wait to place your bags in the beakers until the rest of the class is ready. When the class is ready, place bags in beakers, thus starting the experiment. 7. Mass each bag every 15 minutes. Before massing the bag, blot it gently with a Kimwipe. 8. By reading and recording the mass of each bag at specific intervals, you can determine the rate of diffusion for each treatment. 9. After the last mass data are recorded, analyze and graph your data. First, calculate the percent mass change for each dialysis bag over time. To do this, subtract the initial mass of each dialysis bag (mass @ time = 0 minutes) from the mass for the same dialysis bag at times = 15, 30, 45, and 60 minutes, and divide these values by the initial mass of the dialysis bag. To make this a percentage, multiply by 100

14

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES For example, if the initial mass of the bag is 80 grams at 0 minutes, and the mass at 30 minutes is 90 grams, then the percent mass change is calculated as follows:

Fig. 1. Dialysis bag setup. Experimental Design After you have decided on an experiment to perform as a group and received instructor approval, record the following in your lab notebook: Objective, Variables, Hypotheses, Prediction, Control and Treatment groups, and Procedure. . Refer to the lab notebook guidelines (pages 97 – 99). You will also want to record your data in a well-organized table, which should include times and recorded masses for each bag.

15

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES

Exercise B: Dilution Problems Diluting stock solutions to produce solutions of different concentrations is often necessary in the lab. For example, to make the standard curve (Fig. 2), six standards were prepared from a 150 mg/L starch solution. To perform dilutions we use the following formula:

C1 V1 = C2 V2 where C1 is the concentration of the initial solution, V1 is the volume of initial solution, C2 is the concentration of the final solution, and V2 is the volume of the final solution. Concentration may be expressed in various units, including g/L, %, and molarity (M). Volumes can also be in a variety of units. The important things is to use the same units for both concentration variables, and the same units for both volume variables. The variables for concentration and volume do not have to match – we can use mg/L for concentration and mL volume without converting mL to L, as in the example below. Example: To calculate the amount of 150 mg/L starch solution needed to make 5 mL of a 30 mg/L starch standard, we would do the following: 1. Figure out which variable (C1, C2, V1, or V2) you are solving for and reorganize the equation to isolate that variable. Here, we want to know how much initial solution is needed (V1):

V1 = C2 V2 / C1 2. Next, replace all the known quantities with their values. We know the concentration of the stock solution (150 mg/L), how much standard we want to make (5 mL), and what standard concentration we want (30 mg/L):

V1 = (30 mg/L) (5 mL) / (150 mg/L) 3. As with conversion problems, we can cancel out units and solve the equation:

V1 = (30 * 5 / 150) mL V1 = 1 mL 4. To actually create the standard we would dilute the calculated initial volume to the final volume by adding solvent. In this case, we would add 4 mL of deionized water (our solvent) to 1 mL of 150 mg/L starch solution to produce 5 mL of 30 mg/L starch solution.

16

GENERAL BIOLOGY LAB MANUAL – LAB 3: MEMBRANES With your group, solve the following dilution problems. Record your work in your lab notebook. You must show all of your work to receive full credit for this assignment. 1. How much 150 mg/L starch solution do you need to prepare 3 mL of 60 mg/L starch solution? How much water would you need? 2. If you dilute 15 mL of 200 g/L sugar solution by adding 35 mL of water, what will the concentration of the resulting solution be? 3. If you dilute 175 mL of a 20% sugar solution to 1L by adding H2O, what is the final concentration of the solution?

Clean-up Before you leave lab:  Dialysis bags can be disposed of in the trash.  Rinse all glassware three times. Return all items to the tray on your table.  Make sure you clean your table before you leave. Wipe down as necessary.

Homework Assignment 2: Figure This week you will prepare a figure to represent the data you collected today and submit it to WyoCourses. Refer to the lab report guidelines, example lab report, and lab report rubric (page 101 – 112), and see the assignment description on WyoCourses for more information.

Acknowledgements This lab was written by Diane Gorski and modified by Chris North .

17

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS

LAB 4: MICROSCOPES & CELLS Learning Objectives 1. 2. 3. 4. 5. 6.

Learn to use compound and dissecting microscopes correctly, and name and explain the functions of the parts of these microscopes Calculate magnification properly Estimate the size of objects viewed under the microscope Prepare wet mount slides of living cells to view with the compound microscope Compare and contrast prokaryotic and eukaryotic cells. Compare and contrast plant and animal cells.

Pre-lab Reading

From Textbook (Freeman et al. 2014): Sections 7.1 & 7.2 The cell is the basic structural and functional unit of all living organisms, whether they are singlecelled or multicellular. Learning about cell structure will help you to understand the complexity of cell functions. Microscopes remain an invaluable tool for studying the structure and function of cells and organisms. They are historically important in allowing many advances in science. In this lab you will use two types of light microscopes, the compound light microscope and the dissecting (or stereoscopic) microscope, to examine the structure of different cell types. Cells: Characteristics and Types All organisms are composed of cells. Some organisms, such as bacteria and protists, are composed of a single cell. Other organisms are multi-cellular and may be composed of trillions of cells! It has been estimated that the human body contains between 15 and 70 trillion cells. At the most fundamental level, there are two types of cells: prokaryotes and eukaryotes. The word prokaryote means “before kernel”, and refers to the fact that prokaryotic cells lack a nucleus (the “kernel” or “karyon”). Organisms from two of the three domains of life, Bacteria and Archaea, are prokaryotes. On the other hand, eukaryotes (“true kernel”) are organisms whose cells contain a nucleus, which contains the chromosomes (bundles of DNA) of eukaryotic cells. Eukaryotes include protists (a variety of single-celled organisms with nuclei) and multicellular animals, plants, and fungi. All eukaryotes belong to the domain Eukarya (sometimes called Eukaryota). Aside from the presence or absence of a nucleus, prokaryotes and eukaryotes differ in a number of other ways. One of the first differences you will notice as you examine cells under the microscope is that most eukaryotic cells (5 – 100 μm in diameter) are much larger than prokaryotic cells (1 – 10 μm in diameter). Another difference is that, in addition to the nucleus, eukaryotic cells contain membrane-bound organelles while the vast majority of prokaryotic cells do not (until recently, it was thought that no prokaryotes had organelles). Membrane-bound organelles are small internal compartments within cells that perform specialized functions. Examples include mitochondria and chloroplasts. During this lab, you will compare prokaryotic cells to eukaryotic cells. You will also compare cells from two different types of eukaryotes, plants and animals. Two major differences between plant and animal cells that you should be able to observe with the microscope are the cell wall and chloroplasts. Plant cells and almost all prokaryotic cells possess tough exterior cell walls composed of carbohydrates that protect and support cells, while animals lack cell walls. Chloroplasts are organelles possessed by plants (and eukaryotic algae) that allow these organisms to collect light 19

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS energy from sunlight and store this energy chemically in sugars. Along with mitochondria (another type of organelle), chloroplasts are believed to have originated by endosymbiosis, the incorporation of once free-living bacteria into the eukaryotic cell. One piece of evidence that supports endosymbiosis theory is the fact that chloroplasts and mitochondria each contain their own circular chromosome, like prokaryotes. On the other hand, the nucleus of eukaryotic cells contains multiple, tightly-coiled, long strands of DNA. The chromosomes of eukaryotes become visible under the microscope during cellular division, a process we will observe later in the semester. Anatomy of the Microscope: The Compound Microscope Before lab, familiarize yourself with the anatomy and use of the microscope.

Fig. 1. Basic anatomy of a compound microscope. Descriptions of each component on the following page.

20

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS Base: The base supports the microscope. Lamp (Illuminator): The source of the light that passes through the specimen. Rheostat: The Rheostat dims or brightens light from the lamp. Coarse Adjustment (Focus) Knob: The coarse focus knob moves the stage up and down. This adjustment is used only with the low power objective lens (4×. Fine Adjustment (Focus) Knob: The fine focus knob moves the stage up and down to precisely focus the specimen. This is the only knob you should to focus with the high power objective lenses (10× and 40×). Stage: The stage supports the slide, which is held in place by stage clips, and/or a mechanical stage apparatus. Condenser Lens: The condenser lens is located under the stage and focuses the light on the specimen. It should remain at the highest position close to the stage. Objective Lenses: The objective lenses forms the first magnified image of the specimen. The amount of magnification is marked on the side of each lens. Always use the lenses in order, from low to high power, to avoid crushing the specimen and find objects most easily. Lenses are made of very high quality glass, and should be cleaned carefully. During this lab, we will not use the 100× oil immersion objective lens. Nosepiece: Holds the objective lenses. Body Tube: Joins the nosepiece to the eyepiece (ocular). Arm: Supports the body tube. Always have one hand on the arm when carrying the microscope. Ocular Lenses (eyepiece): further enlarges the image that is magnified by the objective lens. The ocular lenses on the microscope you are using magnify 10X, and are binocular (two eyepieces) as opposed to those with one eyepiece (monocular).

21

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS Understanding the Microscope Setup and Storage: Turn the rheostat all the way down before turning the light on or off to extend the life of the light bulb. If the cord is wrapped around the base, fully unwind it before use. The cord should never be wrapped around the base while the microscope is in use. Using the microscope while the cord is wrapped around the base can damage the gears within that allow the stage to move up and down to focus. Before leaving, make sure the lenses and stage are clean (remove any slides). The low power objective should be in place. If your laboratory instructor instructs you to do so, wrap the cord around the base, cover the microscope with the plastic cover, and put it in its numbered position in the microscope cabinet. Magnification: Magnification of a specimen makes it appear larger than actual or life size. The amount of magnification is designated by a number followed by an “×,” which stands for “times life size.” To determine the total magnification of an object, multiply the magnification of the objective lens by that of the ocular. For example, a 40× objective used in conjunction with a 10× ocular lens yields a total magnification of 400×. Resolution: Resolution is the amount of detail that can be seen in a specimen. Magnifying an object and enhancing the contrast of the specimen (often by staining or manipulating the light passing through the specimen) increase the amount of observable detail. In addition, the care with which a specimen is prepared, and the quality and alignment of the microscope lenses affect the resolution of detail. Generally, the condenser should be in the highest position (close to the stage) with the iris almost completely open for optimal resolution. Partial closure of the iris may at times increase contrast, but if it is closed too much, the image deteriorates. Illumination: The compound and dissecting microscopes require adequate light to create an image of the specimen. To regulate the amount of light, use the rheostat in the compound microscope, and the lamp and mirrors in the dissecting microscope. Do not change the condenser height of the iris opening (this will reduce image quality). To prevent the bulb from burning out, the rheostat must be turned down before the lamp switch is turned off. Viewing: Adjust the distance between the binocular eyepieces as necessary. Objects must be mounted on clean glass slides and usually are covered with (disposable) glass cover slips. The cover slip protects the objective lens, and protects the specimen. Focusing: Always focus first with the low power lens (4×) on the compound microscopes, using the coarse focus knob before the fine focus adjustment. Then, the high power objectives (10× and 40×) may be positioned and focused, using only the fine focus knob. We will not use the 100× oil immersion objective in this class. Cleaning: Use only lens paper or other materials specifically provided by your instructor to clean the ocular and objective lenses. Do not use facial tissues, paper towels, or your fingers. If necessary, clean the stage with Kimwipes and 70% ethanol.

22

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS

Exercise A: Anatomy and Use of Light Microscopes In this lab you will be use a compound microscope for higher magnifications and a dissecting microscope for lower magnifications to examine a variety of biological specimens. As you work through this exercise, draw what you view through the microscope and record answers to questions in your lab notebook. For Drawings, be sure to include the total magnification, a title, labels, and notes as appropriate. Always remove microscopes from the cabinets carefully by holding the arm of the scope with one hand, resting its base on your other hand. Keep the microscope upright as you carry it to your table. Before you turn on your compound microscope, identify its parts using the diagram (Figure 1) and descriptions above. Use of the Compound Microscope 1. 2. 3. 4.

5.

Obtain a letter “e” microscope slide. Use stage clips to hold the slide in place on the stage. Use the knobs below the stage to move the clipped slide so that the ‘e’ is centered over the opening. With the lowest power objective lens in place, use the coarse focus to move the stage up so that the objective is close to the slide (1-2 mm). Look through the microscope and adjust the iris so that the field of view is sufficiently bright (iris almost completely open for optimal resolution), and adjust light further using the rheostat. Do not adjust coarse/fine focus at this time. Look at the microscope from the side, turn the coarse focus knob until the low power objective lens is just above the slide. The stage will move up and down; the objective lenses are stationary within the vertical plane. Note which way you turned the knob to lower the lens - (towards or away from you). Look through the ocular and turn the coarse focus knob in the opposite direction so that the stage (slide) moves away from the objective lens. Continue until the “e” is in focus. Use the fine focus knob to precisely focus the “e”.

CAUTION: Many microscopes have parfocal lenses, which means that as you switch from one lens to another very little focusing is required after the new lens is in place. If a microscope is not parfocal, as you move from one lens to another the new lens may not have enough room above the specimen and may “crash” into the specimen as the lens is moved into place, damaging the lens and/or specimen. When working with a microscope that is not parfocal it is important to lower the stage slightly before moving from a lower to a higher magnification and watch carefully from the side as you move the higher magnification lens into place. Raise the lens further, if necessary, to allow the lens to swing into place without contacting the specimen. 6.

To observe the “e” at a higher power of magnification (with the 10× objective lens), you will first have to center the specimen in the field of view. This is important because your field of view decreases as you change to a higher power. You may have to adjust the fine focus slightly. If you do not see the “e” after you have

23

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS switched to the next highest power, return to low power, make sure the image is centered, and try again. 7. In your lab notebook, draw the letter “e” as it appears when you look at the slide from the side, not through the ocular. In another drawing, depict the letter “e” as it appears when viewed through the ocular at a total magnification of 100×. 8. Move the slide away from you with the knobs below the stage while observing the ‘e’ through the microscope. Which way does the ‘e’ move (up or down)? Move the slide to the right while observing it through the microscope. Which way does the ‘e’ move (left or right)? Record your answers in your lab notebook. 9. What are the total magnifications available on the compound microscope you are using? Record your answer in your lab notebook. Use of the Dissecting Microscope The dissecting microscope has lower overall magnification than the compound microscope. Like the compound microscope, the dissecting microscope uses ocular and objective lenses to create a magnified image of the specimen. Carry the dissecting microscope as you carried the compound microscope - with one hand supporting the base and the other holding the microscope by the arm. Examine the dissecting microscope and identify the following parts: eyepieces, magnification knob, focusing knob, arm, base, stage, objective housing and mirror. Note that illumination for the dissecting microscope comes from an external light source. Operation of the dissecting microscope is relatively simple compared with the compound microscope – use the magnification knob on the top of the microscope to set magnification, the focus knob to bring the specimen into sharp focus and the external light source to provide illumination of the specimen. 10. 11.

12.

13. 14.

What are the total magnifications available on the dissecting microscope you are using? Record your answer in your lab notebook. Place the letter “e” slide under your dissecting microscope at a magnification of 30X. In your lab notebook, draw the letter “e” as it appears when you look at the slide from the side, not through the ocular. In another drawing, depict the letter “e” as it appears when viewed through the ocular of the dissecting microscope. Move the slide away from you while observing it through the microscope. Which way does the ‘e’ move (up or down)? Move the slide to the right while observing it through the microscope. Which way does the ‘e’ move (left or right)? Record your answers in your lab notebook. Compare and contrast the dissecting and compound microscopes. How are they alike? How are they different? Record your answers in your lab notebook. Review the differences between the two types of microscopes. Consider the basic differences in form and function to consider advantages and disadvantages for each kind of microscope when using these microscopes as a scientific tool. What kind of work or uses may be better suited to the compound microscope? Why? What kind of work or uses may be better suited to the dissecting microscope? Why? Record your answers in your lab notebook.

24

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS Estimating cell size One way of estimating the size of cells or other objects under the microscope is to use the (approximate) diameter of field for each lens. In the example below (Fig. 2), if the diameter of field for the 40X objective is 480 m, what is the approximate size of each cell?

Fig. 2. View of cells at 400X

Table 1. The approximate diameter of field for the general biology microscopes with different objective lenses. Objective lens 4X 10X 40X 100X

Leitz 4800 m 1920 m 480 m 190 m

25

Olympus 4500 m 1800 m 450 m 180 m

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS

Exercise B: Prokaryotic and Eukaryotic Cells In this exercise you will look at prokaryotic and eukaryotic cells under the compound microscope, and view some distinguishing characteristics. You will also examine basic characteristics that differentiate plant cells from animal cells. Prokaryotic Cells Prokaryotes are single-celled organisms that include the bacteria and archaea. You will observe Nostoc, a photosynthetic bacteria (also known as a cyanobacteria or blue-green algae). Nostoc cells are smaller than eukaryotic cells, with diameters ranging from 1 to 10m. 1. Obtain a prepared slide of a Nostoc. Focus it under low power, then move to a higher power lens (40×). You will not use the 100× oil immersion lens during this lab. 2. Once you have identified the Nostoc cells, draw what you see in your lab notebook and answer the following questions: What do you see in the cytoplasm? Can you see a nucleus or any membrane bound organelles? On your drawing, label the cell wall and cytoplasm of a cell. 3. Colonies of Nostoc, which occur in long strings, often contain differentiated cells called heterocysts (literally “different cells”). Heterocysts fix nitrogen, effectively pulling nitrogen out of the air for the cells to use - most other cells must obtain nitrogen from food or through absorption from solution. If you drew a heterocyst, identify it with a label. Eukaryotic Cells Animal Cells: Human Cheek (Epithelial) Cells Make a wet mount of your own cheek cells: 4. Place a small drop of distilled water on a clean slide. 5. Gently scrape the inside of one of your cheeks with the blunt end of a flat toothpick, and stir the cells into the distilled water on the slide. 6. Add a small drop of methylene blue stain, and a cover slip. Slowly lower the coverslip over the mounting solution and specimen. If necessary, gently tap on the surface of the coverslip to remove air bubbles. 7. Focus the cells under low power, then high power. 8. Sketch and describe the cells in your lab notebook. Label the cell membrane, nucleus, and cytoplasm. 9. Using Table 1, estimate the size of your cheek cells and add this to your drawing. 10. Can you see small dots or rods in the area surrounding the cells or speckled over the cell surface? What might these small structures be? Record your answers in your lab notebook. Plant cells: Elodea (an aquatic plant) 11. Remove one of the smaller leaves from the tip of an Elodea sprig in the aquarium, and prepare a wet mount. 12. Focus the leaf under the compound microscope and examine one of its cells. Draw the Elodea cells in your lab notebook, and label the cell wall, cell membrane, nucleus, cytoplasm, and vacuole(s) of one of the cells. 13. What are the small, roundish, green organelles? What is their function? Label them in your drawing. Record your answers in your lab notebook. 26

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS 14. Using Table 1, estimate the size of an Elodea cell and add this to your drawing. 15. Next, prepare two more Elodea wet mounts. To one, add a few drops of 9% salt solution. To the other, add a few drops of distilled water. Before looking at the Elodea cells on each slide, predict what you expect to see. Think back to last week. Now look at the cells. What do you observe? Record your answer in your lab notebook.

27

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS

Exercise C: Viewing Aquatic Organisms Freshwater ponds and streams, and aquariums often have an abundance of interesting and diverse single-celled and multicellular organisms. You may not be able to distinguish subcellular components, but you can try identifying some of the more common organisms found in the local waters.

Prepare wet mounts from the sample water(s). Your instructor may offer hints for finding

organisms within each sample. Many of the organisms are visible swimming about in the water. Other good places for finding a diversity of organisms is near any plant material in the sample or along any bottom material in the sample. If you don’t find much on your first attempt, try making another slide, but sample from a different part of the jar. You should use both the compound and the dissection microscopes to examine the water sample organisms. Wet mounts with coverslips must be used for samples examined under the compound microscope, but you may use wet mounts, depression slides or culture dishes for examining specimens under the dissecting microscope. As the specimens in this exercise are not stained, different methods for improving contrast in the specimen will be employed. Experiment with adjusting the light passing through the specimen by slowly closing down the iris or adjusting the rheostat. Focus on low power and examine the organisms. Move up to higher power objectives if you wish. If the organisms are moving too quickly, move to a clean slide and add a drop of Proto-slo to slow them down. As directed by your instructor, sketch some of the organisms. With your sketches, include the magnification of your image and any additional notes on movement, color, shape, structures, interesting features and identification of your organism(s).

28

GENERAL BIOLOGY LAB MANUAL – LAB 4: MICROSCOPES & CELLS

Clean-Up Before you leave lab:  Make sure you clean your table before you leave.  Remove any slides from the stage. Return depression slides, and prepared Nostoc and letter ‘e’ slides, to where they came from.  Place used coverslips and any broken microscope slides in the “Glass Disposal” or “Sharps Disposal” containers – do NOT place these items in the trash cans.  Wash and rinse the microscope slides you used for wet mounts. These can be reused.  Put the lowest power objective lens in place on your compound microscope, turn the rheostat to its lowest setting, and turn off the light.

Homework Assignment 3: Scientific Literature 1 This week you will find a peer-reviewed scientific journal article, learn how to cite it properly, and practice paraphrasing. See the assignment description on WyoCourses for more information. Your submission will contain two files: (1) A pdf copy of the article you found, and (2) a second file with your citations and paraphrasing.

Acknowledgements This lab was written by Diane Gorski and modified by Chris North.

29

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES

LAB 5: MACROMOLECULES & ENZYMES Learning Objectives 1. Understand the difference between quantitative and qualitative methods to compare and measure the concentration of a solution with colorimetric techniques. 2. Know how a spectrophotometer is used to make quantitative measurements 3. Gain experience working with solutions 4. Apply colorimetric techniques to assess the function of an enzyme (amylase) under control and treatment conditions. 5. Learn how to prepare an introduction for a scientific paper. 6. Practice dilution problems.

Pre-lab Reading

From Textbook (Freeman et al. 2014):

Bioskill 3 – Reading Graphs (black-edged pages) Sections 8.1 and 8.3

Macromolecules and Enzymes In lecture, you have discussed the four major classes of organic molecules: proteins, nucleic acids, carbohydrates, and lipids. In this lab we will work with a carbohydrate (starch) and a protein (the enzyme amylase). Carbohydrates are molecules composed of the elements carbon (C), hydrogen (H), and oxygen (O). You can remember this because the –hydrate part of carbohydrates refers to water (H20), and many carbohydrates are actually composed of some multiple of CH2O. For example, the molecular formula of sugar (glucose), a molecule we will encounter in this lab and throughout the semester, is C6H12O6 (6 × CH2O). Carbohydrate molecules play important structural roles (cellulose is the main component in wood), and they are also used by cells to identify one another. Many carbohydrate molecules, such as starch in plants and glycogen in animals, are important for energy storage as they can be broken down into sugar for quick use. Today, we are going to do just that: break starch in to its constituent sugar molecules. But just how are we going to break down starch? Chemical reactions may occur spontaneously, some with dramatic effect, but many occur slowly or do not occur at all without help. In biological systems, help comes in the form of enzymes. Although proteins may have other functions, and are also important for tasks such as defense and movement, enzymes are arguably the most important form proteins take. An enzyme is a catalyst, which means it speeds up (or catalyzes) a specific chemical reaction. For example, amylase breaks down starch and is found in the saliva of many animals. Enzymes are usually easy to identify from their name, as they often end in –ase. Usually the name also tells you something about the particular enzymes function. Starch is actually composed of two components, amylose and amylopectin, so it makes sense that amylase works on starch. The material that is worked on by the enzyme, starch in this example, is called the substrate. Enzymes work by helping to speed up (catalyze) reactions in two ways: (1) they bring substrates together, and (2) they lower activation energy. Enzymes are proteins, often have complex shapes, and are great examples of how form and function are connected in biology. Enzymes have special regions called active sites that can bind to specific substrates. These active site function to bring substrates together, or expose substrates to 31

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES conditions which allow the substrates to break down more easily. Reactions require a certain amount of energy to get started, which is termed the activation energy of the reaction. By changing form when attached to substrates, enzymes can produce conditions under which the reaction requires less activation energy. When less activation energy is required, reactions occur more readily and quickly. Because enzymes have very specific functions, they also tend to work best under specific conditions. For example, pepsin (a protease which breaks down proteins in the stomach) in the stomach (which is fairly acidic) works best in low pH environments. When enzymes are exposed to conditions that are less optimal they function more slowly or may not function at all. This occurs because proteins start losing their form in a process called denaturation when conditions cause molecular bonds to break. The Spectrophotometer and Colorimetry A common tool used in biology and chemistry to analyze the transmission or absorbance of light through materials is the spectrophotometer (Fig. 1). Spectrophotometers are composed of two main components: (1) A light source that can be set to a specific wave length, and (2) a device that can measure the amount of light. The first component, the spectra part, usually consists of a light bulb, a prism, and a slit. By moving the slit or the prism, the wavelength of the light can be selected. The amount of light of the selected wavelength that passes through the sample is measured by a light detector, the photometer part.

Fig. 1. Diagrammatic representation of the main components of a spectrophotometer. Visible light is the part of the electromagnetic spectrum with wavelengths of between 400 and 700 nanometer (nm = 10-9 m). Different colors of light have different wavelengths; red light has a wavelength of about 700 nm, while blue light is closer to 400 nm. When light hits an object, it is either absorbed, transmitted, or reflected. The reflected light is the color we see. For example, when sunlight hits a leaf, green light is reflected, while blue and red light are absorbed by chlorophyll. One of the most common uses for a spectrophotometer is to measure the concentration of a substance dissolved in a solution. This is possible because all substances absorb some light, and if the substance is more concentrated then more light is absorbed, and less light is transmitted through the sample. Sometimes chemicals (called color reagents) can be added to solutions to produce colored compounds when they react with a particular substance. The more of that substance that there is in the solution, the deeper the developed color will be. Methods which use color reagents are referred to as colorimetric techniques. During this lab, you will be using a color reagent called Lugol’s solution (I2KI). Sometimes simply referred to as iodine, Lugol’s solution is used as an antiseptic in medicine and an emergency disinfectant for drinking water. I2KI can also be used to detect the presence of starch because it reacts with starch to produce a dark blue color. The more starch there is in a solution, the darker blue it will turn when iodine is added. When we use a substance like Lugol’s solutions to detect a 32

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES substance, or to compare the relative concentration of a substance in different solutions we are using qualitative methods. However, in science we often want to assign a specific values to measurements. For example, we might want to know how much more starch is in solution A than solution B. With a spectrophotometer and a standard curve we can estimate the concentration of solutions. By using this this method, we might find that solution A has a starch concentration of 113 mg/L, while solution B has a starch concentration of 76 mg/L. Techniques that produce numeric data like this are called quantitative methods.

Fig. 2. Standard curve for starch concentration. Absorbance was measured at 580 nm after 2 drops of Lugol’s solution were added to starch standards. To estimate the concentration of starch in a solution by measuring the absorbance of light with the spectrophotometer, we must first create a standard curve (Fig. 2). A standard curve is created by measuring the absorbance of standards, which are solutions of known concentration. We can compare absorbance values measured from unknown solutions to the standard curve to get an estimate of the concentration of the unknown solution. During this lab, you will use a standard curve and the spectrophotometer to estimate the starch concentration of three unknown solutions. Then you will design and conduct an experiment to assess the effects of some treatment on the activity of amylase. You will also practice dilution calculations. Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. 1. What enzyme will we be working with today, and what is its function? 2. How do enzymes work to speed up chemical reactions? 3. What factors might affect enzyme activity and the rate of chemical reactions? 4. Do you understand the difference between transmission, reflection, and absorbance of light? 5. Can you differentiate between qualitative and quantitative methods? 6. Given an absorbance value, for example 1.002, can you estimate the starch concentration by using the standard curve represented by Fig.2?

33

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES

Exercise A: Measuring Concentration with the Spectrophotometer Estimate starch concentrations of the unknown solutions X, Y, and Z. In a well-organized table, record the following in your lab notebook: unknown solution, absorbance measured, estimated starch. Working with Solutions Solutions are homogenous mixtures of a solute (that which is dissolved) and a solvent (that which dissolves the solute). The concentration of a solution is described as the amount of solute over the amount of solvent. Today, you will be working with starch solutions in units of mg/L, in which starch is the solute and H2O is the solvent. When working with solutions, follow these guidelines: 1. Always agitate solutions before sampling from them to insure proper mixing. 2. Use a new pipette tip for each solution you sample. You may use the same tip repeatedly with one solution before changing the tip. Plan accordingly. 3. Draw liquid slowly and carefully with the pipette Methodology To measure the absorbance of a solution with a spectrophotometer, use these directions. With all methodological directions, it is wise to read through the entire process before starting. 1. Turn on the spectrophotometer (“spec”) and allow it to warm up for a few minutes. 2. Set the spec to 580 nm. 3. Run a blank. A blank is a solution which does not contain the compound that is being estimated, but contains all other components of the experimental solution. We are estimating the concentration of starch. 4. Remove the blank. 5. Obtain 5 mL of an unknown solution. 6. Add 2 drops of I2KI to develop color. Cover and invert to insure the solution is well mixed. 7. Record the absorbance of the unknown. With the standard curve (Fig. 2), estimate the concentration of starch by adding the color reagent (I2KI).

34

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES

Exercise B: Enzyme Experiment Question: How does some factor affect the enzyme activity of amylase? During this experiment, you will design and carry out an experiment at your table. Your objective is to investigate how some factor affects the rate at which amylase breaks down starch in solution. A rate expresses change over time, and in this case you will examine the rate of decrease in starch as it is converted to sugar by amylase under different conditions. To do this, you will perform a pair of time series experiment. When amylase is added to starch solution it immediately begins breaking down the starch. The addition of iodine stops the reaction because iodine reacts with the starch, preventing its further break down by amylase. By adding amylase first, then adding iodine after various time intervals, you can estimate the rate at which starch is broken down. This reaction happens quickly, and a maximum time interval of 120 seconds is appropriate for this experiment. Starting Questions Discuss the following questions with your group. You may want to record your responses on a white board. 1. What are some factors you think might affect the rate at which amylase breaks down starch? Select one of these factors to investigate. 2. What are the dependent and independent variables for your experiment? What are your hypotheses? 3. What will your blank be for this experiment? Will you need different blanks for your control and treatment series? Examine the Methodology on the next page. 4. If you don’t use an experimental solution in your treatment series, what should you put in its place? 5. Design an experiment to test these hypotheses. Get approval from your lab instructor before starting the experiment. Available Materials Starch solution (150 mg/L) Amylase solution (0.5 g/L) Lugol’s solution (I2KI) Spectrophotometer

Test tubes and test tube rack Micropipette and pipette tips (P-1000; 100-1000 μL) Water bath and ice, for heating and cooling Acid and base solutions, for adjusting pH

35

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES Methodology To measure the effect of some factor on the enzyme activity of amylase, you will perform a timeseries experiment with a control series and a treatment series. Here is a basic methodology that you will alter for your own experiment 1. Prepare a blank and blank the spec. 2. Prepare a series of control tubes. For a basic setup, include the following in each tube: - 2 mL starch solution (150 mg/L) - 2 mL deionized H2O - 0.5 mL experimental solution (optional acid or base solution) 3. One tube at a time, add 0.5 mL of amylase (start reaction), wait a predetermined amount of time, then add 2 drops of I2KI (stop reaction). 4. Record the absorbance for each control tube. 5. Repeat for the treatment series.

Fig. 3. Diagrammatic representation of an enzyme time series. Amylase is added to start the reaction, then 2 drops of I2KI are added to end the reaction. However, for the 0 s time, I2KI should be added first, so that amylase cannot react with the starch. Blank tubes have purposely been left empty – what should be included in blanks for each series?

36

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES Experimental Design After you have decided on an experiment to perform as a group and received instructor approval, record the following in your lab notebook: Objective, Variables, Hypotheses, Prediction, Control and Treatment groups, and Procedure. Refer to the lab notebook guidelines (pages 97 – 99). You will also want to record your data in a well-organized table, which should include times and measured absorbance for control and treatment series.

37

GENERAL BIOLOGY LAB MANUAL – LAB 5: MACROMOLECULES & ENZYMES

Clean-Up Before you leave lab:  Turn of the spectrophotometer. Make sure you have not left a sample in the spectrophotometer.  Empty your test tubes, triple rinse them, and return them to your test tube rack upside down so they can dry. Place a paper towel under the tube rack.  Dispose of used pipet tips in the glass waste bin.  Clean the whiteboard if you used one.  Tidy up anything else at your table as necessary.

Homework Assignment 4: Introduction This week you will prepare an Introduction for your lab report on the plant growth experiment. When providing background information, you will need to reference some of the scientific papers you and your classmates located. As you do this remember to paraphrase and cite papers appropriately (see lab report guidelines). See the assignment description on WyoCourses for more information.

Acknowledgements This lab was adapted from a series of labs presented in: Jordan, C.N., and C.A. North. 2014. Life 1010 General Biology Lab Manual. Hayden-McNeil, Plymouth, MI.

38

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION

LAB 6: FERMENTATION Learning Objectives 1. Understand the process of fermentation. 2. Design and conduct an experiment to test how some factor may affect the rate of fermentation by yeast. 3. Evaluate and interpret data collected from a single treatment experiment with a t-test.

Pre-lab Reading

From Textbook (Freeman et al. 2014): Sections 9.1 & 9.6 Cellular Respiration and Fermentation All cells require energy to perform the chemical reactions that allow them to maintain homeostasis (control internal conditions), grow, and reproduce – in essence, live. Cells use energy in the form of adenosine triphosphate (ATP) to fuel these chemical reactions. ATP has high potential energy and is unstable, so it is produced by the cell continuously from other molecules, especially the sugar glucose (C6H12O6). This is accomplished by cells through two main processes, cellular respiration and fermentation. Cellular respiration is a set of controlled chemical reactions that oxidizes glucose to release energy, which is in turn used to produce ATP from adenosine diphosphate (ADP) by adding a phosphate group. This occurs in four main steps (glycolysis, pyruvate processing, citric acid cycle, and electron transport and oxidative phosphorylation – see Fig. 9.2 in Freeman), and produces approximately 29 ATP per glucose molecule, as well as carbon dioxide and water. However, cellular respiration requires a final electron acceptor, usually oxygen. When the final electron acceptor is not available, many cells use fermentation instead to continue producing ATP. You are likely familiar with fermentation as the process by which yeast (a fungus) is used to produce alcoholic beverages such as beer and wine. The production of carbon dioxide by yeast during fermentation is also used to leaven bread. You might also know that fermentation during anaerobic activity, when lack of oxygen begins to limit respiration by your cells, produces lactic acid which can cause cramps and nausea during intense exercise. Although we often associate fermentation with alcohol, lactic acid, and carbon dioxide, these are by-products – the main function of fermentation is to regenerate NAD+ (an electron acceptor) from NADH so that glycolysis can continue. Glycolysis, the first step of cellular respiration, produces 2 ATP from glucose and requires NAD+. Glycolysis also produces pyruvate, which is further processed during cellular respiration to generate more ATP. However, when an electron acceptor such as oxygen is lacking, cellular respiration cannot continue. Instead, fermentation regenerates NAD+ so that glycolysis can continue to produce ATP. Glycolysis occurs in the cytosol, the fluid within a cell but outside of the membrane-bound organelles, while the other three steps of respiration occur within the mitochondria. During lab today you will design and perform an experiment to test how some factor may affect the rate of fermentation by yeast (Exercise A).

39

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Data Analysis in Biology For many scientific experiments and observational studies, the goal is to compare groups (such as control and treatment groups) and determine if there are differences between these groups. If an experiment is well-designed, we can attribute differences to our manipulation. For example, if our plant experiment shows differences in growth between our control and treatment group, we have good reason to suspect that our treatment (e.g. nutrient addition) had an effect. How do we determine if there are differences between groups? This lab presents some of the basic tools biologists and other scientists use to see if differences exist between groups, and provides an exercise to practice using these tools. Scientists often use averages and standard deviations to describe measurements from different groups, and use statistical tests, such as the t-test, to detect differences among two groups. Exercise B demonstrates how to use Excel to calculate averages, standard deviations, and perform t-tests. The exercise also provides several examples to practice calculating and interpreting these basic statistics. Averages and Standard Deviations The most basic way to compare groups is to calculate averages for each set of measurements or observations. For example, let us consider the following hypothetical data from two similar, but independent, experiments (Table 1). We will compare plant height (in cm) data from each of the separate experiments which are similar to the plant growth experiments that form the basis for your semester lab report. Table 1. Hypothetical plant height (cm) data for two independent experiments to illustrate interpretation of data with averages and standard deviation. Experiment 1 Control Treatment 5.6 6.7 4.4 7.9 5.5 8.5 4.9 7.3 5.3 8.2

Experiment 2 Control Treatment 4.6 7.7 4.4 7.9 9.5 8.5 2.9 4.3 4.3 10.2

If we calculate the average for each group, we find that in both experiments plants in the treatment group are taller than plants in the control group (treatment average = 7.7, control average = 5.1 cm). Therefore, at first glance we might conclude that our treatments resulted in taller plants in both experiments. However, we must remember that we have only sampled a handful of plants (n =5), and random factors may have contributed to these differences - for example, maybe the controls were planted poorly, or we happened to choose better seeds for our treatment groups. Another way to compare data is to look at the variability in the data. Standard deviation is one measure we can use to look at variability. Standard deviation essentially measures the dispersion of data around the average – in other words, how close are data points to the average? If standard deviation is low then data points are close to the average, and random factors probably had less of an effect. If standard deviation is large then data points are further from the average, and random factors probably had a greater impact. If we calculate the standard deviation for each group in our hypothetical experiments (Table 1), we discover that data from Experiment 2 is more variable (Table 2). 40

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Table 2. Averages and standard deviations of plant height (cm) for a set of hypothetical experiments. Experiment 1 Experiment 2 Control Treatment Control Treatment Average 5.1 7.7 5.1 7.7 SD 0.5 0.7 2.5 2.1 The greater variation in experiment 2 suggests that random factors have a bigger impact on our results. As a result we might be less inclined to believe that the difference in averages among groups represent a real effect of the treatment. One way to look at this data is to plot the averages with standard deviations (Fig. 1).

Figure 1. Averages and standard deviations (n= 5) for control and treatment groups from two different hypothetical experiments (experiments 1 and 2). Remember, standard deviations are a measure of variance around the average. To compare standard deviations between groups visually, we often add error bars to that represent the average ± standard deviation. For experiment 1, the standard deviations for the control and treatment groups do not overlap, so we might conclude that the difference in averages is a result of our treatment (Fig. 1). For experiment 2, the standard deviations do overlap, which suggests that the difference in averages are more likely a result of random factors (Fig. 1).

41

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Introduction to Statistics: The t-test What is statistics? Broadly speaking, statistics is the study of techniques for analyzing data. The various statistical techniques available to researchers are often called tests or models. In general, the goal of a statistical test is to determine the probability (or chance) that differences detected in data are the result of random chance, and do not represent real effects. What is a t-test? The t-test is one of the simplest statistical tests. It is used for comparing averages between two groups (such as a control group and a treatment group) to determine if they a different. How does a t-test work? A t-test essentially compares the averages and variability of two groups of measurements. The major output of a t-test (and many other statistical tests) is a p-value. You do not need to worry about how this is computed at the moment – you will likely take a statistics class at some point during your college career and learn about the underlying math in more depth. What is a p-value? A p-value is a number between 0 and 1 that represents the calculated probability that a difference between samples is due to random chance. If the probability that an observed difference is due to chance is large (i.e. a larger p-value), then we cannot be confident that the difference is real. If the probability that an observed difference is due to chance is low (i.e. a smaller p-value), then we can be more confident that an observed difference is due to a real treatment effect. How are p-values interpreted? Our choice of a cut-off for a p-value is somewhat arbitrary. However, most scientist use a p-value of 0.05 as their cut-off (this arbitrary cutoff is called the alpha level). Essentially this means that we’re willing to accept a 5% probability that a difference is due to random chance and doesn’t represent a real difference. A p ≤ 0.05 is interpreted as a statistically significant difference. In terms of null and alternative hypotheses, if we find a significant difference (p ≤ 0.05) between our control and treatment groups, we can reject the null hypothesis and accept the alternative. We conclude that our independent variable did have an effect on our dependent variable. If we don’t find a significant difference (p > 0.05), we cannot reject the null hypothesis. We therefore conclude that our independent variable has no effect on the dependent variable. How are t-tests performed? A t-test can be performed by hand, but nowadays people use computer software packages to conduct statistical tests. There are also many web-based statistical tools. We will use Excel, as it is fairly simple to perform a t-test in Excel, and it is available to all students in the University computer labs.

42

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. You do not need to record your answers in your lab notebook, but you may find it useful. 1. What are the four steps of cellular respiration? Where does each step occur within the cell? 2. Which step of cellular respiration also occurs during fermentation? 3. How many ATP molecules are produced from a single molecule of glucose during cellular respiration? During fermentation? 4. What are the reactants and products of fermentation by yeast? 5. What are some factors that you think may affect the rate of fermentation by yeast? 6. How is a t-test used to determine if a statistically significant difference exists between groups?

43

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION

Exercise A: Fermentation Experiment In your table groups, design and perform an experiment to test how some factor may affect the rate of fermentation by yeast. To measure fermentation you will use an apparatus consisting of a 50 mL centrifuge tube with a Luer lock in the cap. A 25 cc syringe is screwed to the Luer lock. Each group will receive four fermentation apparatuses. You will add 25 mL of yeast solution, prepared by your lab instructor, to each experimental tube. You may also add any of several available substrates (e.g. glucose, corn starch, table sugar, and soy isolate). A typical amount of substrate to use is 0.5 g, but you may adjust this. For each treatment or control group you should have at least 3 replicate measurements. Each replicate should run for 20-30 minutes. Starting Questions Discuss the following questions with your group. You do not need to record your answer in you lab notebook, but you may want to record your responses on a white board. 1. How will you measure fermentation with the apparatuses provided to you? (Hint: Think about the reactants and products of fermentation) 2. What are some factors that could affect the rate of fermentation? 3. For this experiment you might want both a negative and positive control. If you were to decide to test the efficiency of a substrate, such as corn starch, what would you use for negative and positive controls? 4. Design an experiment to test one factor with the materials provided. Be sure to consider controls and replication. Get approval from you lab instructor before proceeding. Available Materials Fermentation apparatus (four per group) Digital balances and weigh boats Yeast solution Water bath (~ 40 °C) and ice, for heating and cooling

Beakers and graduated cylinders Digital timers Substrates

Methodology 10. Obtain approximately 100 mL of prepared yeast solution from your lab instructor. Add 25 mL of yeast solution to each tube. 11. Add substrate to the centrifuge tubes as required for your experiment. 12. Screw on a lid without a Luer lock and syringe to each tube and vortex until well-mixed. 13. Carefully remove the first lid (some pressure will have formed) and clean the lip of the tube with a Kimwipe so the tube will form a tight seal with the second lid with a syringe attached. 14. Before screwing on the lid with the syringe, pull out the syringe slightly so that it is set at 1 mL. This will allow the piston to move more easily as gas is produced. 15. When you screw on the lid with the syringe, be careful to handle only the lid and not the syringe so as to not break the epoxy seal. You also should not overtighten the lid. This begins the experimental times. 16. Record the volume of gas in the syringe every 5 minutes. 17. Repeat as necessary to produce enough replicates. Rinse tubes thoroughly between replicates.

44

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Experimental Design After you have decided on an experiment to perform as a group and received instructor approval, record the following in your lab notebook: Objective, Variables, Hypotheses, Prediction, Control and Treatment groups, and Procedure. Refer to the lab notebook guideline, and record your data in a well-organized table. Once you have started your experiment, begin to work on Exercise B in pairs.

45

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION

Exercise B: Statistical Analysis with the t-test

This exercise explains how to perform a t-test in Excel and provides some example exercises. You may use your own laptop or one of the lab laptops to complete this exercise in pairs. How to calculate averages and standard deviation, and performs a t-test, with Excel 1. Enter your data into two different columns, one for your control and the other for your treatment. For this example we’ll use the data from our 1st hypothetical plant experiment (Table 1). Add labels to identify where averages and standard deviations will be calculated, and where you will perform a t-test (calculate a p-value).

2. In the cell next to the label Average, type ‘=AVERAGE(’. The equal sign tells Excel that you want it to perform a calculation (use a function). Next, highlight the first column, then close the ). Excel will return the average.

46

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Next, calculate the standard deviation using the =STDEV() function. Again, highlight the first column of data (but not the average). Calculate averages and standard deviations for both groups.

3. To perform a t-test, we’ll use the function =TTEST().Anytime you start typing a function, Excel will show you what the required fields are. For a TTEST, you need to identify array1 and array2 (which are the two groups you wish to compare), define the number of tails, and the type of t-test. To fill the array fields, highlight the data you wish to use as before. Type a comma after selecting the first array, and before selecting the second array. For our purposes, use a 2-tailed t-test (so, enter ‘2’ for the tails field), and enter ‘3’ for the type.*

* t-tests may be one-tailed or two-tailed, and refer to the extremes of a normal (bell curve) distribution. One-tailed tests are used when we consider differences between groups only possible in one direction (group A must be greater than group B), whereas two-tailed tests are used when either direction is possible (A may be larger or smaller than B). Excel can perform three different types of t-tests: paired (type 1), equal variance (type 2), and unequal variance (type 3). Paired t-tests are used with experimental designs that include paired sampling, such as when an individual is measured before and after some treatment. To determine if variance is equal or not additional statistical tests are required. Without performing these tests it is safer to assume that variances are unequal because the type 3 test is more conservative (less likely to produce false significant results). Similarly, two-tailed tests are more conservative than one-tailed tests. Feel free to adjust the tails and type to see how this affects the p-value.

47

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION 4. Once you have filled all the fields and hit enter, Excel will return a p-value. In this case, our p-value is 0.0003, which is less than 0.05. Therefore, we can conclude that the groups are significantly different. In other words, our alternative hypothesis that our treatment did have an effect on plant growth is supported; the treatment made plants grow taller.

Practice Problems Use Excel to calculate averages and standard deviations, and perform t-tests. Record answers to the questions below in your lab notebook. 1. Perform a t-test for Experiment 2 (Table 1). What is the p-value you get? How would you interpret this result? 2. In fall 2012, students in lab section 21 had an average height of 66.6 inches, while the average height in lab section 17 was 64.6 inches. Here are the data: Student Height (inches) Section 21: 69, 70, 66, 63, 68, 70, 69, 67, 62, 63, 76, 59, 62, 62, 75, 62, 72, 63 Section 17: 68, 62, 67, 68, 69, 67, 61, 59, 62, 61, 69, 66, 62, 62, 61, 70 * Example adapted from the Handbook of Biological Statistics

a. b.

What are the null and alternative hypotheses for this comparison? Perform a t-test on this data. Are the average heights of the two sections significantly different? Explain.

48

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION 3. A researcher suspects that the lateral habenula, part of the vertebrate brain, is involved in appetite suppression. She predicts that electrical stimulation of the lateral habenula will result in decreased food intake in rats. To test this idea, electrodes are implanted in the brains of 20 rats. Following a ten-day recovery period, the rats were randomly divided into two groups and offered chocolate chips during a ten minute feeding period. The treatment group received electrical stimulation during the feeding period, while the control group received no stimulation. Here are the data: Chocolate chips consumed by experimental rats Treatment Control group group 9 12 7 9 3 7 5 14 8 8 5 7 4 12 7 6 7 5 4 8 a. b. c. d.

What are the dependent and independent variables for this experiment? What are the null and alternative hypotheses for this experiment? What is the average and standard deviation for each group? Perform a t-test on this data. Based on the outcome of your t-test, and the values you calculated for c, what is your interpretation of the results of this experiment?

4. Once you have completed your fermentation experiment, calculate averages and standard deviations, and perform a t-test to compare the amount of CO2 produced in control tubes and treatment tubes – if you have multiple treatments, use only one for this comparison. Once you have done calculations and performed a t-test, fill in the blanks in the paragraph on the next page, which demonstrates how a results section might be written.

49

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION Results On average, control tubes produced _____ ± _____ cc of CO2 over _____ min (n = _____ ), while tubes in the _______________ treatment group produced _____ ± _____ cc of CO2 over the same period of time (n = _____ ) (Fig. 1). A t-test comparing the control and treatment groups indicated that _______________________________ (p = _____ ).

A figure representing fermentation data

Fig. 1. Production of CO2 by fermentation in control (n = _____ ) and _______________ treatment tubes (n = _____ ) over _____ min. Carbon dioxide production was ______________________________________________________ (p = _____ )

50

GENERAL BIOLOGY LAB MANUAL – LAB 6: FERMENTATION

Homework Assignment 5: Data Analysis and Results This week you will prepare a Results section for your lab report on the plant growth experiment. Before writing your results section you should analyze your data for significant differences between your control and treatment groups with a t-test. Your Results section should include both visual (i.e. figures) and written representations of your results. See the assignment description on WyoCourses for more information, and refer to the lab report guidelines, example lab report, and lab report rubric (pages 101 – 112).

Clean-up Before you leave lab: 1. Wash centrifuge tubes and any glassware you dirtied (e.g. graduated cylinders). Dry and return tubes to your table and glassware to wherever you got it. 2. Return syringes to the side shelf unless they have been dirtied. Dirty syringes can be discarded. 3. Wipe up any spilled yeast solution with a sponge or wet paper towel.

Acknowledgements This lab was written by Chris North.

51

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS Learning Objectives 1. Understand the reactants and products of the light dependent and independent reactions of photosynthesis. 2. Use a spectrophotometer to characterize the absorption spectra of plant pigments. 3. Graph these data to prepare an absorption spectrum for each solution. 4. List and describe the principle classes of plant pigments.

Pre-Lab Reading

From Textbook (Freeman et al. 2014): Chapter 10 (p. 176-191) Photosynthesis Photosynthesis is a series of endergonic reactions in which plant and algae collect energy from sunlight and store it as chemical energy. The process of photosynthesis in eukaryotes occurs in organelles called chloroplasts, and is typically regarded as occurring in two steps: the light dependent reactions and the light independent reactions. During the light dependent reactions (the photo- part of photosynthesis) pigments, especially chlorophyll, capture energy from photons, which is used to generate ATP and NADPH. During this process water is split to provide electrons for reduction of NADP+ to NADPH, and to produce a proton gradient for ATP synthase. Oxygen (O2) is released as a byproduct. The light dependent reactions occur in thylakoids, sac-like structures within chloroplasts. The light independent reactions (the –synthesis part of photosynthesis) are sometimes called the dark reactions, but they actually occur during light conditions in most plants, at the same time as the light dependent reactions. During this part of photosynthesis, ATP and NADPH produced by the light reactions are used to reduce carbon dioxide to sugars for energy storage, a process known as carbon fixation. ATP and NADPH are oxidized to ADP and NADP+ and returned to the light dependent reactions. The specific set of reactions that make up the light independent reactions are called the Calvin cycle. The Calvin cycle occurs in the stroma, the space inside the chloroplast, but outside the thylakoids. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (commonly called RuBisCO) is a key player in the Calvin Cycle and serves to capture carbon dioxide in the first step. Sugars produced by photosynthesis are used by plants for various purposes, and provide energy to animals that consume them. Ultimately, almost all energy in biological systems is derived from sunlight via photosynthesis. Plant Pigments Plants possess distinctively colored substances known as pigments. Most substances absorb certain wavelengths of visible light (Table 1) more than other wavelengths, and reflect what is not absorbed. For example, if a substance is placed in visible light and absorbs all wavelengths except yellow, then the yellow light is either reflected off of the substance or is transmitted through it. This substance appears yellow to us, as we see the wavelengths of light which are not absorbed by the material. The types and functions of pigments in organisms are diverse. Some pigments are important for the wavelengths of light which they absorb. For example, photosynthetic pigments in plants such as chlorophyll absorb energy from sunlight. In animals, the skin pigment melanin absorbs light and protects other tissues from damage by high energy wavelengths (i.e. ultraviolet light). Other pigments are important for the wavelengths which they do not absorb. These pigments reflect or 53

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS transmit certain wavelengths and give organisms specific visible colors (Table 1). These pigments may be critical in finding a mate, hiding from a predator, or attracting a pollinator or seed disperser. Table 1. Colors and associated wavelengths of the visible spectrum. Color violet blue green yellow orange red

Wavelength (nm) 380-450 450-495 495-570 570-590 590-620 620-750

In this lab you will use the spectrophotometer to gather absorbance data on plant pigments across a series of wavelengths to produce an absorbance spectrum for three different samples (Table 2). First you will need to extract the pigment from your sample. The solvent used to extract pigment will depend on the pigment of interest (Table 2).

Table 2. Extracted sample used in the Photosynthesis and Plant Pigments lab, solvent for extracted sample, and wavelength for initial dilution check. If absorbance is above 2.0 for check, sample must be diluted. Pigments to be extracted Chlorophylls (green and greenish-yellow) Anthocyanins (red, purple, blue) Carotenoids (red, orange, and yellow)

Solvent Acetone

Dilution Check 440 nm

Water

380 nm

Ethanol

400 nm

Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. You do not need to record your answers in your lab notebook, but you may find it useful. 1. What are the reactants and products of photosynthesis? What is the purpose of photosynthesis? 2. What are the two main sets of reactions that occur during photosynthesis? How are they linked? 3. What are some of the various ways that organisms use pigments? 4. If an object appears to be red, what wavelengths are being reflected? Which are likely being absorbed? How about an object that appears green? 5. If you are extracting red pigments from a flower, what solvent should you use? At what wavelength should you do a dilution check? See Table 2.

54

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

Exercise A: Photosynthesis Study Exercise Work with your group to answer the following questions. Record your answers in you lab notebook. 1. What is the equation for photosynthesis? Identify the following components in the equation with labels: source of carbon and oxygen, source of electrons and hydrogen, source of energy for photosynthesis, product of photosynthesis that stores energy, gas produced by splitting of water. 2. Copy and fill in the following table: Stage of Location in Photosynthesis Chloroplast Light Dependent Reactions

Reactant Molecules

Product Molecules

Summary of Stage

Light Independent Reactions (Calvin Cycle) 3. Explain what is accomplished by photosynthesis. 4. Draw a diagram that illustrates the processes of photosynthesis (i.e. a road map of photosynthesis).

55

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

Exercise B: Absorbance Spectrum of Spinach In this exercise you will obtain a spinach extract sample from your lab instructor. With this sample, you will measure the absorbance of spinach at 20 nm intervals from 380 nm to 720 nm. The primary pigment in spinach leaves is chlorophyll a, which absorbs light during photosynthesis and gives leaves their green color. Record the wavelength and absorbance readings in a wellorganized table in your lab notebook. Methodology 1. Turn on the spectrophotometer and allow it to warm-up for 5-10 minutes. The display should have an “A” on the right edge. If it does not, press the [A/T/C] button until the “A” is displayed. The spectrophotometer is now set to display absorbance for all readings. 2. Your instructor will provide each table with a test tube with 5 mL of spinach extract and a second tube with 5 mL of acetone. Do NOT open either of these test tubes. The first test tube is labeled S (spinach), and the second tube is labeled BA (blank acetone). 3. Perform an initial absorbance check on your spinach solution at 440nm: a. Adjust the wavelength to 440nm b. Place the blank in the sample chamber c. Press the [0 ABS] button to blank the spectrophotometer d. Remove the blank e. Place the spinach sample into the spectrophotometer f. If the absorbance reading is above 2.0, notify your instructor. Samples with absorbance values above 2.0 must be diluted. 4. Take absorbance readings every 20 nm from 380 nm to 720 nm (i.e. 380, 400, 420, etc.) The spectrophotometer must be blanked each time you change to a new wavelength. The steps to take a reading are: a. Adjust the wavelength b. Place the blank in the sample chamber c. Press the [0 ABS] button to set the blank d. Remove the blank e. Place the spinach sample into the spectrophotometer

56

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

Exercise D: Absorbance Spectrum of Red Cabbage Prepare a red cabbage extract and measure its absorbance at the same wavelengths as you did for spinach. The primary pigments in red cabbage are anthocyanins (water-soluble red, purple, and blue pigments) that are prized by nutritionists for their antioxidant qualities. Preparation of Red Cabbage Extract: Obtain approximately 15 g of red cabbage – record the exact amount in your lab notebook. Place the cabbage into a mortar, add 25 mL deionized water, and thoroughly grind the cabbage with the pestle for 3 min. Decant (pour off) the liquid into a clean 50 mL beaker and then pour 10 mL of this solution into a labeled 15 mL centrifuge tube. Bring this to your lab instructor so that the raw extract can be further purified by centrifugation. What is the concentration of the extract you prepared? Record your calculations and answer in your lab notebook. Methodology Put 5 mL of the purified cabbage extract in one test tube and label it. Put 5 mL of deionized water in another test tube use as a blank. Follow the same methodology used in Exercise A. If the absorbance is above 2.0, dilute the extract until the absorbance at 380 nm is less than 2.0. Record any dilutions and your data in a well-organized table.

57

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

Exercise E: Absorbance Spectrum of a Selected Sample Obtain one of the various samples available to you and prepare an extract with water or ethanol (see Table 2 to determine which solvent to use). Available samples include various flowers and produce. During the fall semester available samples may also include fall leaves. Measure the absorbance of your extract at the same wavelengths as you did during the previous two exercises. The primary pigments that produce yellow, orange, and red colors in plants are carotenoids, while darker reds, purples, and blues are usually produced by anthocyanins, as in cabage. Carotenoids are sometimes accessory pigments that help chlorophyll collect light energy while also protecting chlorophyll from damage caused by excessive light. What is the concentration of the extract you prepared? Did you have to perform any dilutions? Record any calculations you perform and your data in your lab notebook.

Exercise F: Plot Your Data Plot the data you collected in Exercises A – C, either by hand in your notebook or with Excel. If you use Excel, send a copy to your lab instructor. You may plot three separate graphs, or you may plot all the data on a single figure. Be sure to include a figure legend or figure legends (if you create multiple figures) – a caption that explains what the figure shows. Answer the following questions in your lab notebook: 1. What pigments did you examine today? Based on the absorption spectra you plotted, what colors does each pigment absorb and reflect (refer to Table 1)? 2. What function does each type of plant pigment perform?

58

GENERAL BIOLOGY LAB MANUAL – LAB 7: PHOTOSYNTHESIS & PLANT PIGMENTS

Homework Assignment 6: Discussion This week you will prepare a discussion section for your lab report on the plant growth experiment. Refer to the lab report guidelines, example lab report, and lab report rubric (pages 1010 – 112). Be sure that you cite references to support you interpretation of your results, to make comparisons to other studies, or to suggest modifications. See the assignment description on WyoCourses for more information.

Clean-up Before you leave lab: 1. Return the spinach extract and acetone blank to your lab instructor if you haven’t yet. 2. Ground produce and flowers can be discard in waste bins. Water and ethanol-based extracts and blanks can be dumped in the sink. 3. Rinse any glassware you used thrice (e.g. mortar and pestle, beakers, tubes, etc). Dry and return to your table. Remove any labels. 4. Make sure spectrophotometers are turned off. 5. Do any additional cleaning that is necessary.

Acknowledgements This lab was designed by Diane Gorski and modified by Chris North. Exercise A was adapted from a study sheet produced by Pearson Education.

59

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA

LAB 8: DNA Learning Objectives 1. Understand how polymerase chain reaction is used to amplify DNA 2. Learn how gel electrophoresis is used to analyze DNA samples 3. Use DNA gel electrophoresis to solve a murder mystery

Pre-Lab Reading

From Textbook (Freeman et al. 2014):

Bioskill 9 – Separating and Visualizing Molecules (black-edged pages) Section 15.3

Restriction Enzymes Restriction enzymes were discovered by Werner Arber, Hamilton Smith and Daniel Nathans in the mid- 1970’s. Their discovery revolutionized biochemistry and molecular biology. Most prokaryotes possess protective enzymes that chop the DNA of invading viruses into fragments. The cell’s own DNA is not cut by its restriction enzymes because the cell protects the recognition sites on its DNA by camouflaging them with methyl groups. The restriction enzymes therefore do not recognize these sites on the host cell’s DNA. However, foreign DNA lacks the protective methyl groups on the recognition sites, and thus the bacteria’s restriction enzymes can cut the foreign DNA. Restriction enzymes are highly specific specialized molecular scissors – they cut DNA very precisely at sites with specific base pair arrangements (Fig. 1). Molecular biologists take advantage of these enzymes to cut DNA at specific locations for a variety of purposes. For example, scientists can take a piece of DNA from one organism and insert it into the DNA of another organism using restriction enzymes and ligases to cut and paste, respectively. When DNA is digested (cut into pieces) by one or more restriction enzymes, scientists can use information gathered about the sizes of the different pieces to learn about the original piece of DNA. Sometimes the information confirms that a recombinant molecule has been correctly constructed, other times the sizes of DNA fragments from different sources are used to determine if these sources are similar or related.

Fig. 1. Sample restriction enzyme recognition sites. 61

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA Gel Electrophoesis DNA that has been cut into pieces with restriction enzymes can be separate by size using gel electrophoresis. During gel electrophoresis, DNA is loaded into wells in an agarose gel, then subjected to an electric field. Because DNA has an overall negative charge it will move towards the positive electrode (cathode), and away from the negative electrode (anode). The smallest pieces travel the greatest distance from the starting point. Pieces of the same size travel the same distance. If enough strands of the same size fragment of DNA are present, this group of fragments will show up as a band when the gel is viewed. DNA ladders are collections of DNA fragments of known sizes which are used to determine the size of unknown fragments. The unit used to measure the size of DNA fragments is base pairs (bp) – the number of paired nucleic bases that make up the fragment. Another common unit, used for larger DNA fragments, is the kilobase (kb = 1000 bp). In this lab we will use a DNA ladder (Fig. 2) as a standard to estimate the size of DNA bands in a set of samples to solve a murder mystery.

Fig. 2. DNA ladder used in general biology DNA mystery lab. (MassRuler™ Express Reverse DNA Ladder Mix)

62

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA The Biology of a Murder: The Mysterious Case of the LIFE 1010 Murder On Tuesday, all of the TAs and professors spent the night grading the last exam, and Mr. North (the lab coordinator) stayed late to input the exam grades into his computer. Little did he know that he would spend his last moments entering grades. The next morning Mr. North was found stabbed to death on the floor of his office. He had been stabbed 15 times in the neck with a P-1000 micropipettor, and the killer had left the murder weapon in his victim. An autopsy later revealed dried skin and blood underneath Mr. North’s fingernails, suggesting he had fought his assailant. On that same fateful morning, one of the LIFE 1010 professors, Dr. Willford, was found lying on the floor in the lobby of the STEM building, injured and in shock. In the days that followed, Dr. Willford was interviewed by police at his hospital bedside. He told officers that he suspected Mr. North had been killed by another professor, Dr. Currano, who he had seen leaving the labs as he was returning from the grocery store. He said Dr. Currano had pushed him down the stairs, and that he had been knocked out tumbled. He had laid helpless on the ground until one of the graduate students found him in the morning. Dr. Willford had suffered a severe concussion, a badly bruised right hip, and abrasions to his legs and right arm from the assault. There had been a spate of attempted professor slayings around campus thought to be motivated by the stress of midterms, and Dr. Currano was also.a suspect in those cases. In the room where the grading and murder took place, police found no signs of forced entry and nothing of value had been taken. When forensic teams searched the room, they found evidence that blood had been wiped from a cabinet handle and that Mr. North’s body had been moved. Furthermore, the team noticed that someone had saved a copy of the exam grades one hour after the approximated time of death. The UW Police department’s computer forensics team was able to extract the last auto-saved copy of the file and compared it to the final copy. They noticed that several grades had been changed. Police soon questioned Dr. Willford's story. Several people reported that they’d witnessed Dr. Willford and Mr. North arguing loudly about Pokemon, and the Dr. Willford had threatened to “make him pay”. Also, Dr. Willford had claimed to have been at the grocery store getting snacks for the poor, starving graduate students at the time of the murder. But, although it had snowed that night, a police officer noticed the ground under Dr. Willford's car was still dry. Dr. Willford quickly became a suspect. But there was a problem with that theory – Stuck in his office for ten years, Dr. Willford had little time for exercise. Police thought it doubtful that such a feeble man could overpower Mr. North, who had a black belt in Krav Maga. A few days later, police apprehended Dr. Currano Police extracted DNA from blood samples collected from each suspect (Drs. Willford and Currano).

63

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA

Exercise A: Gel Loading Practice To examine and analyze the DNA samples collected from the two suspects, you will run the DNA in an agarose gel. Agarose is a highly purified seaweed extract. Each gel has a series of holes, called wells, along one edge of the gel, which serve as a loading site for the DNA samples. The gel is submerged in buffer in the electrophoresis device, which sets up the electric field. The electrical field pulls the DNA through the gel, separating out the migrating fragments of DNA based on size. Starting Questions Discuss the following questions with your group. Record your answers in your lab notebook. 1. What is the overall charge of DNA molecules? 2. Will DNA travel from the negative electrode to the positive electrode, or vice versa? 3. Will shorter or longer fragments travel faster? Will shorter or longer fragments be closer to the loading wells? Successfully loading the DNA samples into the tiny wells in an agarose gel takes some practice. You will first practice with small pieces of gels made of non-nutrient agar. These gels are similar to the gels you will use for gel electrophoresis, but do not contain the chemicals necessary for running and viewing DNA samples. At your table you will find materials for this practice session: petri dishes, pipets, loading dye tubes, and a beaker for tap water. The loading dye is a mixture of bromophenol blue dye and glycerol, which are also mixed with DNA to load a sample into a gels. The loading dye serves two important functions – glycerol is denser than water and helps the sample sink into the wells, and the blue dye allows us to monitor the DNA sample’s progress through the gel in the electrophoresis unit. Methodology To practice loading samples into the wells of a gel, follow these instructions: 1. Place a dry practice gel into a petri dish and practice loading the blue loading dye into the wells. Place the end of the pipet near the bottom of the well and fill the well slowly from the bottom up. 2. Next practice with the gel submerged in tap water: Place the gel in the petri dish and cover the gel with tap water. Once again, practice carefully filling the well from the bottom up. Try not to introduce any air bubbles into the well as you add the loading dye. Remove the pipet carefully, keeping the plunger depressed so as to not remove or disturb the loading dye. 3. When finished with your practice gels, place the used gels in the labelled waste container at the sink in the back of the room, rinse out the petri dishes and beakers, and return the practice gel supplies to the tray on your table.

64

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA

Exercise B: Gel Electrophoresis of DNA Samples Your TA will provide your group with four 0.6 mL microcentrifuge tubes, each containing 0.7 μL of either a DNA ladder or the DNA samples collected from the suspects (X = Currano, Y = Willford) and the evidence collected from underneath Mr. North’s fingernails (E). Each piece of evidence was amplified at two different loci (plural of locus, a location or place on a chromosome), labelled 1 and 2. Wear nitrile or vinyl gloves when loading the DNA samples and handling the buffer or agarose gel, or any items which may have come in contact with the gel or buffer. The agarose gels contain ethidium bromide, a mutagen. Small amounts of ethidium bromide can also dissolve into the buffer from the gel, so do not touch the buffer solution without gloves. If you spill the buffer in your electrophoresis unit your TA will help to safely dispose of the spilled solution and refill the buffer to the correct level. Your TA will place the gel in the electrophoresis unit. The gel should be oriented so the wells are to the right (the negative electrode side of the electrophoresis unit). Use the P-20 micropipettor to load the DNA samples (7 µL each) into wells. Use a fresh, sterile tip to load each sample. In your lab notebook, record which sample is loaded into each well by drawing a diagram. Make this drawing large enough that you can also record the band patterns you observe when you view the gel. You should also record the voltage and amount of time used to run the gel. When the gel is done running, use the ladder (Fig. 2) to estimate the DNA fragment size for each band.

65

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA

Exercise C: PCR Group Activity To obtain large amounts of DNA for use in gel electrophoresis and other applications, DNA must be amplified (or multiplied). Polymerase Chain Reaction (PCR) is a laboratory technique which allows researchers to amplify DNA. Amplification may be general or very specific. When amplifying a specific sequence to isolate DNA from a particular loci or gene, specific DNA primers, which bind at both the 5’ end and the 3’ end of the gene, are added to the PCR mix. These primers define the region to be amplified. The DNA amplification process consists of three steps: (1) Denaturation of DNA: high heat is used to separate the DNA strands; (2) Annealing: temperature is adjusted to allow the primers to bind to the DNA template; (3) Elongation: temperature is adjusted again so that new DNA molecules can be polymerized on the template DNA. These three steps are repeated numerous (20+) times in a thermal cycler (or thermocyler), a machine which adjusts the temperature in a predetermined time sequence. Each time, the DNA is further amplified. Answer the following questions on this sheet. 1. For the DNA molecule below, which primer locations (A, B, C, or D) would need to be selected in order to amplify the highlighted gene? 5’ 3’ ATCGTATGTGCAGGGTCGATTTGCGTAACGTTGCAGTTGCAGTTTGACGAGTA A

B

TAGCATACACGTCCCAGCTAAACGCATTGCAACGTCAACGTCAAACTGCTCAT 3’ 5’ 2. Write the 5’ to 3’ sequence for both of the primers which would be required to amplify this gene via PCR.

3. Compared to the DNA replication process within a cell, the denaturation step achieves the activity of which enzyme(s)?

4. Compared to the DNA replication process within a cell, the primer annealing step achieves the activity of which enzyme(s)?

66

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA 5. What enzymes and other materials need to be include in the PCR mix in order for amplification to occur?

6. Diagram 3 cycles of PCR amplification for the gene shown above using the selected primers:

67

GENERAL BIOLOGY LAB MANUAL – LAB 8: DNA

Clean-up Before you leave lab: 1. Dispose of any practice gels at your table in the waste container at the back sink. 2. Discard your gloves in the indicate waster container. 3. Wash your hands before leaving the lab. TA clean-up: 1. Dispose of ethidium bromide gels in the container in the hood 2. Return the spatula and gel transport trays to the viewing stations 3. Wipe down viewers and leave open to dry

Homework Assignment 7: Scientific Literature 2 For your homework this week, you will read a scientific article chosen by your instructors, and write a 1-page synopsis. We will form groups and discuss the article during a lab period. We have you write the synopsis before lab so that you read and try to understand the paper ahead of our discussion. The article and instructions on how to write your synopsis are provided on WyoCourses.

Acknowledgments This lab was written by Diane Gorski and modified by Chris North. The PCR group activity (Exercise C) was contributed by John Willford.

68

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS

LAB 9: MITOSIS AND MEIOSIS Learning Objectives 4. Diagram and describe the major similarities and differences in the processes of mitosis and meiosis. 5. Understand the functions of mitosis and meiosis in organisms. 6. Compare and contrast mitosis in animal and plant cells by examining microscope slides. 7. Relate the process of meiosis to genetics.

Pre-Lab Reading

From Textbook (Freeman et al. 2014):

Sections 12.1, 12.2, and 13.1 Bring your textbook to lab this week, you will find it helpful.

Cell Division One of the tenets of cell theory is that all cells come from pre-existing cells. How does this occur? In all organisms, cells are produced by cell division, during which a parent cell divides into two or more daughter cells. Cell division is the final step of what is known as the cell cycle, essentially the lifetime of a cell. During the cell cycle, cells grow and accumulate resources during a series of gap phases (G1 and G2). Cells also duplicate their DNA during the synthesis phase (S) in preparation for cell division when all of their resources, including genetic information, will be divided among daughter cells. In prokaryotes, cell division is accomplished by binary fission, which produces two identical cells. Because prokaryotes usually only have a single, circular chromosome, during binary fission the cell simply pulls one copy of the chromosome (which was replicated during S phase) to either side of the cell before splitting. Cell division in eukaryotes is more complicated because genetic material contained in DNA is packaged into a number of chromosomes (for example, your cells each contain 46 chromosomes), which must be carefully sorted and divided. Depending on the function of cell division, eukaryotic cells divide either by the process of mitosis or by the process of meiosis. Mitosis and Meiosis Eukaryotes produce new cells through two types of cell division: mitosis and meiosis. Mitosis produces two genetically identical daughter cells, while meiosis produces four genetically different daughter cells. Mitosis produces diploid cells, like most of the cells in your body, which contain two sets (2n) of homologous chromosomes. A pair of homologous chromosomes each has the same set of genes, although they may have different variants of these genes, and one homolog is inherited from each of your parents. On the other hand, meiosis produces haploid cells, which contain only one set (n) of chromosomes. In humans, diploid somatic cells contain 46 chromosomes, while haploid gamete cells contain 23 chromosomes. Before cell division occurs, chromosomes are replicated during the S phase to produce two identical sister chromatids which are separated during division. Diploid somatic (body) cells are created by mitosis. Through the production of somatic cells by mitosis, organisms can grow, heal wounds, or replace old cells. In single-celled eukaryotes, mitosis can also be used for asexual reproduction to make exact copies, or clones, of parent cells. Haploid cells produced by meiosis are the sex cells, called gametes or germ cells. In animals, gametes are eggs and sperm. When two gamete cells fuse during fertilization, they produce a new diploid cell. 69

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS This new diploid cell divides via mitosis, and grows to produce a new organism. Meiosis produces genetically different cells because of independent assortment of chromosomes and crossing over, which will be discussed more during the genetics lab. Because gametes produced by meiosis are each unique and are combined randomly during fertilization, sexual reproduction leads to organisms that are different from their parents. Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. You do not need to record your answers in your lab notebook, but you may find it useful. 1. To insure that daughter cells produced by cell division have the same chromosome complement as the parent cell, what must occur before division starts? 2. What are the five phases of mitosis presented in the textbook? What are the important features of each phase? 3. What is reduction division? 4. How do the products of meiosis differ from the products of mitosis?

70

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS

Exercise A: Mitosis & Meiosis on the Table To better understand the processes of meiosis and mitosis, and the similarities and differences between these two types of cell division, you will model the key stages in each process on your desk. Once you have arrived at the correct model, record your diagram on to the summary sheet at the end of this lab. For this exercise, you will model an organism with 2 chromosomes in its haploid state (n = 2), and 4 chromosomes in its diploid state. Note that this is a simplification because most multicellular organisms contain many more chromosomes. For example, you have 46 chromosomes in your somatic cells. Also, you will not represent crossing over during meiosis in your model. Materials Inventory materials before and after this exercise and let your instructor know if you are missing any pieces or have extras. Do not take apart the paired sister chromatids. 4 lg single chromosomes (3 cm), blue 4 lg single chromosomes (3 cm), red 4 sm single chromosomes (2 cm), blue 4 sm single chromosomes (2 cm), red 4 lg sister chromatid pairs (3 cm with bead), blue 4 lg sister chromatid pairs (3 cm with bead), red 4 sm sister chromatid pairs (2 cm with bead), blue 4 sm sister chromatid pairs (2 cm with bead), red 1 mitosis outline sheet 2 meiosis outline sheets Procedure 1. If you have not already, make sure you have all of the pieces listed above. If you are missing any, or have extras, let your instructor know. 2. Arrange the pieces on the mitosis sheet to show the arrangement of chromosomes and sister chromatids at each stage. When done, raise your hand to have your instructor check your arrangement. For mitosis, you will not need all of the pieces 3. If your instructor tells you that you have the correct arrangement, copy it onto the summary sheet at the end of this lab. 4. Remove the pieces from the mitosis sheet. 5. Arrange the pieces on the meiosis sheet to show the arrangement of chromosomes and sister chromatids at each stage. In your model, you will produce only sperm or eggs, not both. You may choose which to produce. For meiosis, you will need all of the pieces. 6. If you have the correct arrangement, copy it onto the summary sheet at the end of this lab. 7. Return the pieces to their container, making sure they are all there. 8. Now you may help others by giving hints, but do not show them the correct arrangement.

71

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS Follow-up Questions Answer the following questions in your lab notebook: 1. During which stage of the cell cycle does DNA replication occur? 2. Does crossing over occur during mitosis or meiosis? 3. During which stage does crossing over occur? 4. Does mitosis produce somatic cells or gametes? How about meiosis? 5. Most human cells contain a total of 46 chromosomes – how many chromosomes are in haploid cells? How many are in diploid cells? 6. Before going through meiosis, a cell has 12 chromosomes. How many chromosomes do the resulting daughter cells have? 7. Fill in the following table: Mitosis Number of divisions Stage during which homologous chromosomes separate Stage during which sister chromatids separate Number of daughter cells produced Gametes produced

72

Meiosis

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS

Exercise B: Mitosis and Meiosis in Animal and Plant Cells Examine the following slides, and make drawings and answer questions in your lab notebook. Remember to label your drawings and include magnification. Onion Mitosis Slide View a prepared slide of onion (Allium) root tip mitosis under a low-power objective to identify the general shape of cells in this area. The meristematic, dividing tissue is just above the tip of the root. Draw the root tip under low power. At higher magnification, find and draw examples of cells in each stage of mitosis: prophase, metaphase, anaphase and telophase. Fish Mitosis Slide Obtain a slide of mitosis in a whitefish blastula (an embryonic stage). Find examples of metaphase and telophase. Draw these stages and label the structures that differ from those of dividing plant cells (onion mitosis). Lily Anther and Grasshopper Testis Slides When observing meiosis in plants (anthers of Lilium) and in animals (grasshopper testis) keep in mind that meiosis results in four products, while mitosis results in only two. Locate cells that are in the stages of metaphase I or metaphase II in one of these organisms and draw them. How can you tell if cells are in the metaphase I or metaphase II stage?

73

GENERAL BIOLOGY LAB MANUAL – LAB 9: MITOSIS & MEIOSIS

Clean-up Before you leave lab: 1. Make sure all of the materials from Exercise A have been returned to their container. 2. Return slides to their place, lower the stage of your microscope, set it to the low power objective, and turn off the light after turning down the rheostat.

Homework Assignment 8: Peer Review Your homework this week will be to review the discussion section a classmate. Your lab instructor will assign you a discussion section to review. You will use the lab report rubric to grade your classmates discussion, and provide comments and suggestions for improvement. Use the lab report guidelines, example lab report, and lab report rubric (pages 101 – 112) to guide your comments. Does the Discussion section include all of the necessary components? Is each component adequately developed? Is the writing clear and concise? Be sure to give yourself time to complete this task, which must be done before lab.

Acknowledgments This lab was written by Diane Gorski and modified by Chris North. Exercise A is based on an exercise developed by Larry Flammer, who created the mitosis, meiosis, and summary sheets.

74

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

LAB 10: GENETICS Learning Objectives 1. Explore how meiosis and sexual reproduction are connected with genetics. 2. Examine case studies of different types of inheritance and learn how to use Punnett squares to predict the potential outcomes of a cross. 3. Perform a simulation of artificial selection (dog breeding) to investigate heredity and the roll of chance. 4. Develop a pedigree for the results of the dog breeding simulation to better understand the information portrayed by pedigrees.

Pre-Lab Reading

From Textbook (Freeman et al. 2014):

Chapter 14 Bring your textbook to lab this week, you will find it helpful.

Genetics Genetics is a rapidly growing and changing scientific field. The information that previously filled the genetics chapters of biology texts is now a mere scratch on the surface. In this lab you will explore fundamental concepts of Mendelian inheritance. In the nineteenth century, Gregor Mendel established the foundation of genetics with his historic experiments on garden peas. He studied characteristics that varied among individual plants that he carefully bred and followed through many successive generations. Mendel examined characteristics that clearly differed between plants – for example, he looked at purple vs. white flower color, round vs. wrinkled seed coats, and yellow vs. green pods. He also used “true breeding” individuals – that is, his plants with purple flowers had come from plants with a history of purple flowers. Mendel’s studies led him and others to recognize the following: 1. A characteristic or trait, such as flower color, is a heritable feature. (We now call this a gene.) 2. Variations of characteristics occur, for example, purple vs. white flowers. (These variations are now known as alleles.) 3. In individual organisms, alleles occur in pairs; one member of each pair has been inherited from each parent.

75

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

Exercise A: Meiosis and Genetics Answer these questions in your lab notebook. 1. A gene is a segment of DNA which codes for a particular trait. Genes come in varieties, one inherited from each parent. What are these varieties called? 2. What does dominant mean when referring to gene varieties? What does recessive mean? 3. Recall that chromosomes are strands of DNA found in cells. Each chromosome contains many genes. In your lab notebook, draw a circle to represent a eukaryotic cell from a pea plant. Label this circle Figure 1. a) Inside the circle, draw a pair of homologous chromosomes. A single homologous chromosome is called a homolog. b) Next, draw four genes on each chromosome. In this example, the first gene is homozygous, composed of two identical dominant alleles, represented by capital letters. The second gene is homozygous recessive, represented by two lowercase letters. The last two genes are heterozygous, each composed of two different alleles, Rr and Tt. Label each allele (P, g, R, r, T or t). You may use different colors and/or patterns to identify different alleles. If you do, be sure to include a legend. Fig.1 shows one possible way to illustrate these chromosomes:

Fig. 1. A pair of homologous chromosomes with alleles for genes P, G, R and T shown. 4. Gene P, which is homozygous dominant, codes for flower color and results in purple flowers. Gene G, which is homozygous recessive, codes for pea color. Green is dominant, yellow is recessive. What color are the peas that grow on the plant from which this cell came? 5. Gene R codes for pea shape, which can be either round or wrinkled. Gene T codes for plant height, which can be tall (dominant) or dwarf (recessive). What shape are the peas from our example plant? Is our pea plant tall or dwarf? 6. Peas contain seeds. Seeds are produced when a sperm cell (male gamete) and an egg (female gamete) merge during fertilization. Which type of cell division produces gametes?

76

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS 7. Before cells enter mitosis or meiosis they replicate DNA during S phase. In your lab notebook, draw another circle, labeled Figure 2. In this circle, draw the homologous chromosome pair represented in Fig. 1. Remember to include the alleles described in Question 3. Then, draw an identical sister chromatid for each homolog. 8. Recall that chromosomal crossover results in the exchange of genetic material between chromosomes. In your lab notebook, draw another circle, labeled Figure 3, which represents the same cell following crossing over, during Metaphase I (Refer to Figure 13.7 in Freeman and your notes from the Mitosis and Meiosis lab). Remember to include the alleles. 9. At the end of meiosis four gametes are produced, each with half the complement of chromosomes of the diploid organism (Fig. 1) contains. In your lab notebook, draw 4 circles to represent the gametes produced by the division of the cell pictured in Fig. 3. Remember to include the alleles. 10. Describe how meiosis is responsible for the Principles of Segregation and Independent Assortment.

77

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

Exercise B: Punnett Squares and Types of Inheritance Punnett squares provide a convenient method for predicting the results (i.e. expected proportions of offspring by phenotype and genotype) of various crosses. In this exercise, we’ll consider various heritable human traits and diseases, and will use Punnett squares to explore different types of inheritance. First, let’s examine Cystic Fibrosis. Cystic Fibrosis (CF) is an inherited disease in humans that causes the production of abnormally thick mucus linings, affecting the respiratory and digestive systems of inflicted individuals, sometimes leading to deadly lung infections. Cystic fibrosis affects people who are homozygous recessive; individuals with only one recessive allele are referred to as carriers. Homozygous dominant individuals are neither carriers, nor affected by the disease. In this example use F to represent the dominant allele, and f to represent the recessive allele. Answer the following question in your lab notebook: 1. Define the terms genotype and phenotype. 2. A man and a woman are considering having a child. Genetic screening indicates that they are both heterozygous for CF. What are the genotype and phenotype for the couple? 3. Construct a Punnett square. What is the probability that the couple’s child would have CF? If you do not have experience with Punnett squares, see the instructions on p. 262 of your textbook (Freeman et al. 2014). 4. Another couple is also planning on having a child. The man has CF, but his wife is not a carrier. What are the genotypes and phenotypes of the man and woman, respectively? Use a Punnett square to determine (a) the probability that their child would have CF, and (b) the probability that their child would be a carrier. We’ve just examined a typical autosomal monogenic trait, which means the gene involved occurs on an autosome (non-sex chromosome), and involves only a single gene locus (location). Many traits exhibit this type of inheritance, but a number of other types of inheritance are common. Case Studies There are six case studies on the next three pages. Discuss each example with your group and determine how to best represent the type of inheritance with a Punnett square. You may find it helpful to use the white boards to work through problems, but you should record the Punnett squares and answers to any questions for each case study in your lab notebook.

78

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS A. Color Blindness Red-green color blindness is surprisingly common among human males, with 7-10% of men exhibiting color blindness. In women, however, colorblindness is nearly non-existent. Before you consider the genetics involved, let’s examine the mechanism resulting in color blindness. In the human eye there are two basic types of cells that detect light, rod cells and cone cells. Rod cells are sensitive to low light levels, and contribute to peripheral and night vision. Cone cells come in three varieties (blue, green, and red receptors) which are sensitive to different wavelengths of light, and therefore allow us to perceive color. Color blindness occurs when any one of these three types of cone cells are defective. Deuteranomalous red-green color blindness, the most common type of color blindness by far, occurs when the spectral sensitivity of green receptors are altered, such that they are more sensitive to red light than green. When this occurs afflicted individuals have difficulty distinguishing red and green. Such defects are caused by production of faulty proteins in cone cells that detect light. Proteins, as you know, are coded for by genes. It turns out that many of the genes that code for these cone proteins are located on the X chromosome, including those found in green cone cells. In fact, redgreen color blindness is known as a recessive X-linked trait for this reason. When working with X-linked traits, we represent Punnett square crosses between sex chromosomes. In most mammals, and some insects and plants, males carry a Y chromosome and an X chromosome, while females carry two X chromosomes – other organisms have different systems for sex chromosome systems. Typically, we represent X-linked traits by adding superscripts to the X chromosomes in our Punnett squares. For example, Xb could represent the recessive allele that causes color-blindness, while XB could represent the dominant allele. Consider the following: Sally and Tom are planning to have kids. Sally’s father, John, is red-green color blind, but Sally has normal color vision, as does Tom. 1. What can you conclude about Sally and Tom’s genotypes? 2. Use a Punnett square to determine the probability that the couple’s kids will be color blind. 3. How could does X-linked inheritance explain the differences in the occurrence of color blindness in men and women?

B. Eye Color Blue eye color was long believed to be a simple recessive trait controlled by a single gene – that is, an individual with blue eyes must have received a recessive allele from each parent. As a result it was inferred that a child whose parents are both blue-eyed must also have blue eyes. This occasionally led to the erroneous claim that a child with brown or green eyes born to a couple with blue eyes must indicate adultery. We now know that although blue eyes are commonly passed down and all people with blue eyes probably share a common ancestor (Eiberg et al. 2008), eye color is controlled by at least 15 different genes. As a result of this complexity, almost any eye color may result from a pairing of two blue-eyed individuals. Many, probably most, traits are polygenic (controlled by multiple genes) like eye color. Here, for simplicity’s sake, we will consider two important genes leading to blue eyes, the B locus and T locus. Dominance at either locus will result in blue eyes. However, individuals that are homozygous recessive for both genes display brown eyes.

79

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS 1. Use a dihybrid Punnett square cross (see Fig. 14.5 in Freeman et al. 2014) to demonstrate how a couple with blue eyes (both heterozygous for both loci) might produce a child with brown eyes. What is the probability that this couple could have a brown-eyed child? Eiberg, H, J Troelsen, M Nielsen, A Mikkelsen, J Mengel-From, KW Kjaer, and L Hansen. 2008. Blue eye color in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression. Human Genetics 123:177-187.

C. Hair Type Jane is the only person in her immediate family with curly hair. Her parents both have wavy hair, while her two brothers have straight hair. Jane has heard that hair type (curly, wavy, or straight) is inherited and can’t figure out how her type fits in with the rest of her family. Jane’s friend Tori, also has curly hair, but this isn’t surprising because her mother does too. Tori’s father and brother both have wavy hair. Tori has heard that hair type genetics are a bit unusual and display something called incomplete dominance, but neither her nor Jane know exactly what this means. 1. What is incomplete dominance? See page 272 of Freeman et al. (2014). 2. Draw two Punnett squares that show how hair type is inherited in each family (Jane’s and Tori’s) 3. Explain why Jane has curly hair when nobody else in her family does.

D. Blood type I Blood types (A, B, & O) in humans are determined by a set of three different alleles of one gene; one individual can have only two of the three possibilities. Two of the alleles are codominant. The codominant alleles are IA, which is responsible for the production of an A antigen, and IB, which is responsible for the production of a B antigen. These antigens are carbohydrates that are found on the surfaces of red blood cells. The third allele is recessive to both IA and IB and consequently it is designed with a lower case i. The red blood cells of individuals with two i alleles have no antigens on their red blood cells and have type O blood. The phenotypes and genotypes of the four human blood types are summarized below. Type A IAIA or IAi Type B IBIB or IBi Type AB IAIB Type O ii 1. What does this mean, to say that two alleles are codominant? 2. Susie, who has type A blood, and whose biological mother has type B blood, plans to marry Frank, who has type O blood. Give the phenotype and genotype of Frank, Susie, Susie’s mother, and possible offspring of Susie and Frank. What can you determine about the blood type of Susie’s biological father? Explain your inferences using Punnett squares.

80

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS E. Blood type II Mary has type A blood and her biological father, Mike, has type B blood. Jill, Mary’s biological mother, is not certain of her blood type. She thinks she needs to have blood work done to ascertain her blood type. 1. Does Jill need blood work to figure out what type of blood she has? Can her blood type be deduced from the knowledge of Mary’s and Mike’s? Explain and illustrate your response using Punnett squares.

F. Sickle-cell disease Sickle cell disease (SCD, sometimes called sickle cell anemia) exemplifies two genetic concepts: pleiotrophy and heterozygous advantage. Sickle cell disease occurs in people that are homozygous recessive for a gene that produces the blood protein β-hemoglobin. This causes red blood cells, which carry oxygen to different parts of the body, to become misshapen (sickle-shaped rather than disc-shaped as normal blood cells) when oxygen concentrations are low. This shape-change, caused by a single gene, can affect many different traits. The ability of a gene to affect several traits is called pleiotropy. Persons with SCD may suffer any number of chronic or acute conditions, including those that lead to kidney, bone and heart damage, as well as sudden death from various causes. Sickle-cell disease is highly deleterious – that is, it often kills people suffering from it at young ages and should be heavily selected against. However, the allele which causes SCD is more common in areas where malaria occurs, and in people whose ancestors came from these places, than might be expected given the severity of SCD. Malaria is caused by a protozoan parasite transmitted by mosquitoes, and killed more than 650,000 people worldwide in 2010. As it turns out, people who are heterozygous for SCD are more resistant to malaria than either otherwise healthy homozygous dominant individuals or homozygous recessive individuals, who suffer from SCD. This condition, where heterozygous individuals have higher fitness than those with either homozygous phenotype, is called heterozygous advantage. 1. Draw a Punnett square that shows how a single-gene for β-hemoglobin can cause sickle-cell disease (an example of pleiotropy) and confer heterozygous advantage. Use this drawing to explain the two bold terms.

81

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

Exercise C: Probability and Dog Breeding In the previous exercise we examined the use of Punnett squares to predict the outcome of genetic crosses. However, these predictions only represent the probability, or predicted chance, that an offspring will possess a particular trait. In practice, predictions rarely match reality. Answer the following questions in your lab notebook: 1. Consider a gene in dogs that controls tail length. The dominant allele (T) codes for a long tail, while the recessive allele (t) codes for a short tail. Draw a Punnett to predict the ratio of offspring expected to have long tails and short tails given a cross between two heterozygous (Tt) dogs. What proportion of puppies do you expect to have short tails based on your Punnet square? 2. In this exercise, we will use pennies to simulate actual crosses. In each cross you will determine the contribution of each parent randomly by flipping a coin. An outcome of heads indicates the contribution of the dominant allele, while tails indicates the recessive allele. You will perform 20 simulated crosses (i.e. produce 20 simulated puppies). How many of the 20 puppies do you expect will have short tails? 3. Perform the simulation 20 times by flipping a penny twice for each cross. Record the results in your lab note book. 4. How do the results of your simulation compare with your prediction? Historically, much of our understanding of genetics comes from examining breeding in domestic animals and plants. For example, Mendel’s groundbreaking work was performed with pea plants. Nowadays, though, breeders can use knowledge of genetics to improve their results. Of course, random chance still plays an important role. In this final part of this exercise, you and your partner will simulate breeding dogs to better understand the implications of probability in genetic crosses. 5. Choose the traits that you wish to select for each gene (refer to Table 1 on the next page for traits and varieties). For example, you may want produce a long-haired dog breed, with a black coat, upright ears, a short tail, and pale eyes. Record the traits you want in Table 2 in the row labelled Goal Dog. 6. Select one of the ‘starter’ dogs from your TA. What dog did you choose and why? 7. Now enter the information for your starter dog into Gen 1 as mother or father on Table 2. 8. Find a dog belonging to one of your classmates that you can breed with your dog and fill in that dog’s information in Gen 1 as well. Using pennies as you did earlier to determine which allele is inherited, produce 3 offspring from your Gen 1 pairing, and record the genotypes of these offspring in Gen 2. 9. After you have produced three offspring, pick one of the offspring to breed. For the purpose of this exercise, consider hemophilia deleterious (a trait that significantly reduces an organism’s ability to reproduce successfully) - hemophiliac dogs (XhY, or XhXh) cannot be used in the next generation. As before, find a mate for your dog among your classmates, this time from the second (F1) generation. Continue this for 3 generations, filling in the table at each step. 10. Once you have filled in the entire table, assess your results. How successful were you in producing the breed you set out to create? Why or why weren’t you successful?

82

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS Table 1. Traits modeled during a dog breeding simulation. Trait Phenotypes Locus (or Expression Loci) Size Small, medium, A, B Locus A is a growth inhibitor, and dogs dominant or large for this gene but not locus B are small. Locus B is a growth stimulator, and dogs dominant for this gene but not locus A will be large. Dogs dominant or recessive for both loci are medium. A_bb = small A_B_ or aabb = medium aaB_ = large

Ears Hair Length Coat Color

Floppy, or upright Short, or long Black, red, brown, or yellow

F L

An underscore indicates that either allele may be present. Floppy ears (FF or Ff) are dominant over upright ears (ff). Short hair (LL or Ll) is dominant over long hair (ll).

D, E

Tail

Long, or short

T

Eye Color

Pale, grey, or dark

P

D_E_ = black ddE_ = red D_ee = brown ddee = yellow An underscore indicates that either allele may be present. Long tails (TT or Tt) are dominant over short tails (tt). Incomplete dominance: PP = pale Pp = grey pp = dark

83

84

Offspring 3

Offspring 2

Offspring 1

Father

Mother

Gen 2

Offspring 3

Offspring 2

Offspring 1

Father

Mother

Gen 1

Dog Name

Dog Name

Table 2. Dog Worksheet

phenotype genotype phenotype genotype phenotype genotype phenotype genotype phenotype genotype

Goal Dog: phenotype genotype phenotype genotype phenotype genotype phenotype genotype phenotype genotype

Expression

N/A

Locus

B

A_bb = small A_B_ = medium aaB_ = large

A

Size F

Ears L

Hair Length D

E

Coat Color T

Tail P

Eye Color

D_E_ = black Floppy ears ddE_ = red Incomplete Short hair Long tails (F) are D_ee = brown dominance: (L) is (T) are Hemophilia is a dominant ddee = yellow dominant cominant recessive Xover PP = pale An underscore indicates over long over short linked trait, upright An underscore indicates Pp = grey that either allele may be hair (l) tails (t) represented here ears (f) that either allele may be pp = dark present. present with X H or X h

XX = female XY = male

Gender

Trait

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

85

Offspring 3

Offspring 2

Offspring 1

Father

Mother

Gen 4

Offspring 3

Offspring 2

Offspring 1

Father

Mother

Gen 3

Dog Name

Dog Name

phenotype genotype phenotype genotype phenotype genotype phenotype genotype phenotype genotype

phenotype genotype phenotype genotype phenotype genotype phenotype genotype phenotype genotype

Expression

N/A

Locus

B

A_bb = small A_B_ = medium aaB_ = large

A

Size F

Ears L

Hair Length D

E

Coat Color T

Tail P

Eye Color

D_E_ = black Floppy ears ddE_ = red Incomplete Short hair Long tails (F) are D_ee = brown dominance: (L) is (T) are Hemophilia is a dominant ddee = yellow dominant cominant recessive Xover PP = pale An underscore indicates over long over short linked trait, upright An underscore indicates Pp = grey that either allele may be hair (l) tails (t) represented here ears (f) that either allele may be pp = dark present. present with X H or X h

XX = female XY = male

Gender

Trait

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

GENERAL BIOLOGY LAB MANUAL – LAB 10: GENETICS

Exercise D: Pedigrees With the information you recorded in Table 2, produce pedigrees for TWO of the target traits you selected (see your answer to Exercise B, question 5). Include all of your dogs on each pedigree. Represent males with squares, females with circles, and use shading to represent dogs that show the trait in question. Make sure you indicate what trait you are diagramming in each pedigree, and include names for each dog. See section 14.6 of Freeman et al. (2014) for example pedigrees. Write a brief discussion of what each pedigree conveys about the trait it represents. Refer to figures 14.22 and 14.23 in Freeman.

Clean-up Before you leave lab: 1. Return dog cards and pennies to you lab instructor.

Acknowledgements This lab was developed and written by Chris North.

86

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION

LAB 11: EVOLUTION & NATURAL SELECTION Learning Objectives 1. Understand and use histograms to represent three modes of natural selection: directional selection, stabilizing selection, and disruptive selection. 2. Design and conduct an experiment to evaluate a hypothesis about the relationship between variation in traits in a population of isopods and natural selection by a simulated predator. 3. Analyze and interpret experimental data from a natural selection experiment conducted in class with histograms.

Pre-lab Reading

From Textbook (Freeman et al. 2014): Sections 25.3 & 26.3 Evolution & Natural Selection Evolution is one of the defining characteristics of life. In everyday speech, evolution is used to mean change. In biology, evolution has a more specific meaning: change in the genetic characteristics (allele frequencies) of a population over time. A population is a group of individuals of a particular species living in a given time and place. We observe evolution occurring in natural populations, plant and animal breeding, and the development of antibiotic resistance. Through the fossil record, the shared genetic similarities among organisms, and our observations of evolution, we understand that species are related by common ancestry. In his famous book On the Origin of Species, Charles Darwin proposed that natural selection is the main process by which populations evolve. Darwin described evolution by natural selection as the consequence of heritable variation in populations and the fact that not all individuals survive and reproduce. The individuals that are most likely to survive and reproduce are those best adapted to their environment – effectively, well-adapted individuals are selected by nature. As a result, natural selection leads to more individuals in a population with certain heritable characteristics. Although Darwin was very interested in heredity, he did not understand genetics. In fact, the words gene and genetics were not even coined until 20 years after his death. However, given our current understanding of genetics, we can describe the requirements for natural selection in the following manner: 1. Individuals have variable phenotypes. In other words, there is variation in a trait within a population. 2. At least some of that variation in phenotype is due to genotype (alleles). Note that other factors, such as environment, may also affect phenotype. 3. Some phenotypes (individual with certain traits) are more likely to reproduce than others. Phenotypes that are more likely to reproduce have higher fitness. In the biological sense, fitness is equivalent to the ability to reproduce. If these three requirements are met, the allele frequencies of a population will change over time – evolution will occur. While biologists generally accept that natural selection is the primary driver of evolution, other processes may lead populations to evolve. These processes include genetic drift, gene flow, and mutation. When natural selection occurs it may have one of several effects on the distributions of traits in a population. Many traits within a population approximate a normal distribution (bell-shaped curve). For example, consider the distribution of fur color in a mouse population (Fig. 1). Most mice are 87

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION gray, while only a few mice are very light or very dark. Different types of selection may change the distribution of a trait within a population in different ways.

Fig. 1. The distribution of a trait, fur color, in a population of mice. Fur color follows a normal distribution. The vertical line in the center indicates the mean value for the trait. Three types of selection are directional selection, stabilizing selection, and disruptive selection. When individuals with traits at one end of the distribution have the highest fitness, directional selection may occur. When individuals with traits in the middle of the distribution have the highest fitness, stabilizing selection may occur. When individuals at both ends of the distribution have higher fitness than individuals in the middle, disruptive selection may occur. Histograms Histograms are figures that are used to illustrate distribution data (see example Fig. 2). Usually the number of individuals, frequency, or percentage is plotted on the y-axis against some continuous variable on the x-axis. Individual samples or measurements are counted and binned into groups. A bin is a range of values. A well-designed histogram has bins that are (1) adjacent, (2) nonoverlapping, and (3) equal in size. The number of bins used can reveal different features of the data, and is dependent on the number of data points (n). Although there is no set way to decide how many bins to use, one method is square-root choice method in which the number of bins (k) is a function of the sample size (n), where k = √n. However, deciding how many bins to use may require other considerations.

88

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION Consider the example data below for mouse fur color (Table 1). How would we select bins for this data? If we use the square-root choice method we would make 7 bins (√46 = 6.8), but because our data has values between 1 and 16, 8 bins is a better choice (16 is not divisible by 7). Therefore, it is important to consider the range and values of the data in addition to the number of data points when selecting bin sizes. If we use 8 bins for the mouse fur color data, we end up with the histogram shown in Fig. 2. Table 1. Fur color of individual mice (n =46) collected from an abandoned farm field. Color values are on a 16-bit grayscale, where 1 = white and 16 = black. fur color (16-bit grayscale) 11 4 7 12 5 6 10 12 8 10 11 11 10 9 16 3 10 15 8 5 8 2 8 6 5 3 13 9 9 13 6 5 7 9 4 4 12 8 14 14 7 9 10 9 8 7

89

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION

Fig. 2. An example histogram that shows the distribution of fur color among mice (n =46). Color values are on a 16-bit grayscale, where 1 = white and 16 = black. Notice that the histogram above approximates a normal distribution, and that bins are adjacent, non-overlapping, and of equal size. Isopods This week in lab you will carry out a natural selection experiment with isopods. Animals that belong to the Order Isopoda can live in aquatic and terrestrial environments, and may be better known to you as roly-polies (also called wood lice and pill bugs). Terrestrial isopods are often found under decomposing logs and leaves. Isopods belong to the Class Crustacea, and like their marine relatives such as shrimp and crabs, isopods breathe through gills. As a result, they must live in humid environments and do not tolerate desiccation (drying) well. When we work with the isopods in the lab it will be important to keep them moist. We will work with the species Armadillidium vulgare. In nature, A. vulgare play an important role as detritivores - organisms that consume decomposing plants and animals. They are also prey for a variety of predators, including spiders, birds, and frogs. You will investigate natural selection by simulating predation of isopods and measuring and plotting the distribution of some trait before and after predation. Pre-lab Questions You should read and consider the questions below before lab so you are better prepared. You do not need to record your answers in your lab notebook, but you may find it useful. 1. How could you rewrite or paraphrase the requirements for natural selection to occur? 2. Can you draw how the distribution of fur color in mice might change from that shown in Fig. 1 with directional, stabilizing, and disruptive selection? Section 26.3 of Freeman has good examples of these three types of selection. 3. How are bins selected for a well-designed histogram?

90

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION

Exercise A: Histograms and Types of Selection Your lab instructor will assign your table group one of the three example data sets below. Each data set contains measurement from a population of organisms before and after natural selection. To analyze the data, produce two histograms in your lab notebooks, one for the original population and one for the population following natural selection. You may want to combine both sets of data into a single figure, but be sure you can distinguish between the data sets. Remember to follow the guidelines in the pre-lab reading as you prepare your histograms. Your task is to examine the data to determine what type of selection has occurred and present your findings to your classmates on your white board. Data Set 1 Guppies (Poecilia reticulate) are small, tropical freshwater fish that thrive in many habitats. The total length data below (Table 2) came from a guppy population in a small Bolivian headwater (mountain) stream before and after the introduction of a larger, predatory fish. Table 2. Total length (mm) of Bolivian guppies before and after the introduction of pike minnows. total length (mm) before selection 44 42 27 16 31 32 22 15 41 35 42 37 21 29 35 24 30 34 36 25 33 25 36 44 19 23 32 28 30 21 33 39 33 30 26 26 47 41 31 28 26 38 40 39

total length (mm) after selection 17 16 24 19 22 29 43 46 39 49 25 48 46 15 22 17 49 40 45 27 24 20 46 26 34 42 44 20 45 18 20 47 21 30 33 18 22 16 15 47 18 44 36 42

91

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION Data Set 2 American robins (Turdus migratorius) often lay several broods of eggs a year, and each clutch may consist of 2-6 eggs. A group of ornithologists (scientists who study birds) counted eggs in robin nests on a small wooded property in Virginia over the course of several decades. During this time the surrounding countryside was converted from forest to farmland. Table 3 contains clutch sizes recorded in 1983, before agricultural conversion, and data from 2007 after most of the surrounding land became kale farms. Table 3. Clutch size of American robin nests in Floyd County, VA. clutch size (# eggs) 1983 5 3 6 6 6 3 2 3 3 4 4 5 3 2 6 3 5 5 5 5 6 2 5 2 2 3 3 4 4 4 6 4 4 4 4 2

clutch size (# eggs) 2007 3 5 4 2 4 3 4 5 2 2 4 4 3 4 4 4 4 4 4 5 5 4 4 4 3 4 3 6 3 4 3 2 6 5 6 4

92

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION Data Set 3 Plants and pollinators often co-evolve (evolve together). Many moth pollinators use tongue-like proboscises to access flower nectar, and in turn help plants reproduce by moving pollen (male gametes) between flowers. The following data (Table 4) on the length of sphinx moth proboscises were collected during two different years (2010 and 2014). Table 4. Proboscis lengths of sphinx moths collected in southwestern Colorado in late July 2010 and 2014. proboscis length (mm) 2010 11 6 6 8 8 6 8 6 8 9 9 9 8 9 7 10 5 7 10 10 8 9 10 9 8 8 7 8 11 7 8 8 7 10

proboscis length (mm) 2014 8 9 10 9 8 6 13 6 8 11 7 11 8 10 7 10 12 12 9 11 10 10 9 12 7 10 7 11 11 11 8 9 10 10

93

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION

Exercise B: Isopod Natural Selection Experiment For this experiment, you will explore the natural selection with a population of Armadillidium vulgare isopods. We will play the role of predator and compare measurements of an isopod trait (either length, mass, or color) from the whole population to measurements from the survivors after the simulated predation event. Isopod predators include birds and shrews (modeled with forceps), frogs and lizards (modeled with a sticky rod), and spiders and foxes (modeled with a fork). Pick an isopod trait to examine, and a predator to simulate. What kind of selection do you predict will occur, if any? Experimental Design After you have decided on an experiment to perform as a group and received instructor approval, record the following in your lab notebook: Objective, Variables, Hypotheses, and Prediction. Refer to pages 6-7 of the lab syllabus. You will also want to record your data in a well-organized table, and produce histograms to interpret your data. ISOPOD CARE Isopods are living organisms and we want to be careful and considerate when working with live animals to avoid unnecessary harm. Additionally, we will use these isopods throughout the week, so it is important to keep our isopods healthy for other lab sections. Be gentle and try to avoid hurting the isopods – you may want to use paintbrushes to move isopods. Also, because isopods are crustaceans that breathe through gills they should be kept moist – use the Isopod Water spray bottle to wet the soil and paper towel in their container, and avoid leaving isopods under lights for any amount of time. Methodology After selecting a trait, predator, and forming hypotheses and predictions: 1. Get a container of isopods and carefully count them into a large culture dish to insure that you have 40 isopods. Replace any missing isopods from the extras container, or return extras you might have. 2. Get a tool for your simulated predator - forceps for birds and shrews, a glass rod with tape for frogs and lizards, or a fork for spiders and foxes. 3. Setup the simulation arena. Each arena should have four refuges (coffee filter sections), one in each corner, and a ring in the center. 4. Recruit a naïve hunter – a classmate who does not know which trait you are examining. 5. Place all 40 isopods in the ring, then release them. Allow the isopods 60 seconds to scatter before beginning the hunt. 6. The naïve hunter now has 5 minutes to “eat” half of your isopods (20). Be careful not to hurt the isopods – only use the sticky rod on the backs of isopods so they don’t lose legs, and use the fork to scoop, not stab. Also, isopods that make it to the refuges (under coffee filter sections) are considered safe and cannot be captured. 7. Place the “eaten” animals in a labeled small culture dish. 8. After the hunt, find all remaining “survivor” isopods and place them in a labeled small culture dish.

94

GENERAL BIOLOGY LAB MANUAL – LAB 11: EVOLUTION & NATURAL SELECTION 9. Give the “eaten” and “survivor” culture dishes to your lab instructor. He or she will re-label them. 10. Take measurement from both groups. After completing measurements, have your lab instructor identify the two groups. 11. Create a histogram which compares the whole population of isopods (all 40 you started with) to the survivor group. Follow-up Questions After producing histograms of your data, discuss the following questions with your group. Record your answers in you lab notebook. 1. What type of selection, if any, did your observe in your experiment? 2. What is the relationship between variation and selection? For example, the number of dorsal plates on isopods does not vary within species – how do you predict selection might impact this trait? 3. If predation and other selection pressures reduce variation, why does variation persist in nature?

Clean-up Before you leave lab: 4. Return isopods, soil, and paper towels to their container. Return containers to the back bench. 5. Rinse and dry the large and small culture dishes. 6. Return rulers, paintbrushes and any other materials you used.

Acknowledgements This lab was adapted by Chris North from a series of labs presented in Jordan & North (2014), originally based on Berkelhamer (1998): Jordan, C.N., and C.A. North. 2014. Life 1010 General Biology Lab Manual. Hayden-McNeil, Plymouth, MI. Berkelhamer, R. 1998. Variability and selection in natural populations of wood lice. Proceeding of the Association for Biology Laboratory Education 19:245-254.

95

GENERAL BIOLOGY LAB MANUAL – APPENDICES

LAB NOTEBOOK GUIDELINES Lab and field notebooks are essential for all scientists. Your notebook is a record of experiments, data, observations, and even ideas and questions you may come up with while performing an experiment. A well-maintained notebook helps keep data organized and is invaluable when you need to recall what you did. In some disciplines, such as medical research, keeping an accurate laboratory notebook also protects proprietary data and is an important tool for preventing fraud. At several times during the semester you will provide your lab instructor with your lab notebook so he or she can check to make sure you are recording all pertinent information correctly. Completeness will be the most important factor. The following guidelines will help you take good notes.

General Rules 1. On the front cover of your lab notebook include the following: your name, your lab section, course and semester. 2. Dedicate the first page to a Table of Contents (see Organization and Contents below). 3. Because lab notebooks are permanent (and sometimes legal) records of work done, always use a pen. 4. If you make a mistake, draw a thin line through the word or number. Do not obscure the original entry. Sometimes these “mistakes” prove to be important. 5. Similarly, never remove a page from your notebook. 6. Write legibly! 7. Always include appropriate units with all data, and in all figures and tables.

Organization and Contents 1. Enter the page number, lab title, and date in the Table of Contents at the beginning of each lab period. 2. Write the page number, your name, the date, and the lab title at the top of each page. 3. For labs during which you perform an experiment, record all pertinent information as outlined below. 4. For some labs you will be asked to answer Starting and/or Follow-up Questions. Include these and any other information you are asked to include in your lab notebook.

97

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Experimental Information Objective A brief (one or two sentence) explanation of the purpose of the experiment. What question do you want to address? For example, your objective might be to test the effectiveness of a new fertilizer on the production of tomatoes by a tomato plant. Variables The independent variable is the variable you change or manipulate in an experiment. Groups within the independent variable are referred to as treatments. The dependent variable is the effect you measure. It is sometimes also called the response variable. For example, you might want to measure the effect of fertilizer (independent variable) on the tomato production (dependent variable). Your specific independent variables (or treatments) might be the amount or type of fertilizer used, and you specific dependent variable might be number or mass of tomatoes produced. Include both the independent and dependent variables for each experiment. Hypotheses The null hypothesis is the hypothesis that the independent variable has no effect on the dependent variable – e.g. adding the new fertilizer does not affect tomato production. The alternative hypothesis, on the other hand, is the hypothesis that the independent variable has an effect on the dependent variable – e.g. adding fertilizer affects tomato production. Note that the alternative hypothesis does not speculate as to the nature of the effect – this is the role of your prediction. Include both the null hypothesis and alternative hypothesis for each experiment. Prediction If there is an effect of your treatment (e.g. addition of the new fertilizer), what do you expect it to be? For the tomato example, you would probably predict that the addition of fertilizer will increase the production of tomatoes. But you could also predict a decrease in the production of tomatoes.

98

GENERAL BIOLOGY LAB MANUAL – APPENDICES Control group(s) A control group is an experimental group to compare against your treatment group(s). Whenever possible, scientists aim to compare experimental treatments to both a negative control and a positive control. A negative control is an experimental group for which no effect is expected. In the tomato experiment example, a negative control might be a group to which no fertilizer is added, or a substitute that should have no effect (inert glass beads, for example) is added in place of fertilizer. Without a negative control to provide a baseline, it is not possible to determine whether the new fertilizer actually increases tomato production. A positive control is an experimental group for which an effect is expected. In the tomato experiment example, a positive control might be a group to which a proven fertilizer is added (that is, a fertilizer we know should increase tomato production over baseline). If the positive control doesn’t show an effect this suggest something else is wrong with the experiment – for example, we may not have added enough water and for this reason none of the plants did well. It is not always possible to design negative and positive controls – positive controls in particular can be difficult. Imagine, for instance, an experiment to test the effect of temperature on tomato production. No positive and negative controls are readily apparent. Instead, we might simply have a control group at room temperature (if the experiment is performed in the lab). Our experimental groups could then be tomato plants grown under heated and/or cooled conditions, and tomato production in the experimental groups could be compared to the room temperature control group. Procedure Your procedure should be described with sufficient detail that another person could perform your experiment based on these notes alone. You will also use your procedures as the basis for the Methods sections of lab reports, so you want to be sure to include all important information, including sample sizes and replication. It is easiest to record your procedure as a list, and leave spaces between entries so you can make notes or edits as necessary. It is not necessary to copy the entire methodology presented in lab handouts. However, you will want to refer to these methodologies and make note of any change you made. Data You should record data in organized tables with all units clearly indicated. You may also be asked to sketch figures of your data on occasion. Plotting data in a figure can be a good way to visualize interesting trends or relationships between variables.

99

GENERAL BIOLOGY LAB MANUAL – APPENDICES

LAB REPORT GUIDELINES

You will produce an individual lab report based on an experiment you carry out as a class over multiple weeks. Lab Reports mimic the style scientists use when publishing the results of their work in peer-reviewed journals. We have you write a lab report to help you: 1. Develop a better understanding of the scientific process which is synthesized in scientific articles. 2. Become acquainted with methods for analyzing and interpreting data 3. Become familiar with the style in which scientific findings are reported. 4. Improve your ability to write clearly and concisely Below you will find helpful guidelines for preparing your lab report. You should also regularly refer to the lab report rubric as you complete homework assignments and prepare your lab report. An example lab report is also provided.

Parts of a Research Paper Title Titles should be informative with both the independent and dependent variable given. Abstract Note: You are not asked to include an abstract for your lab report, but you should be aware that they are important elements of most research papers. Abstracts provide: 1. An overview of the entire project/paper 2. One to two sentences each of introduction, methods, results, and conclusions 3. Do not refer to literature, statistics, or tables/graphs 4. Should be the last section written Introduction 1. Background Information o A review of prior literature that is relevant to understanding the rest of the paper  Highly audience specific  Must cite sources in-text (see below) 2. Purpose Statement and Hypotheses o Explain why the research was conducted o Give research question and objective o State the null and alternative hypotheses. o Provide a prediction of expected outcomes

101

GENERAL BIOLOGY LAB MANUAL – APPENDICES Methods 1. Written in past-tense (work has already been performed), and not in second person (i.e. “you”) 2. Include the following in methods o study area/organisms used o study design, including  description of controls  sample size  replication o sampling and measurements used o statistics used 3. Often divided into subsections to aid in organization 4. Report details in a logical topic based manner – not in order conducted 5. Do not give a list of procedures (step-by-step instructions) Results 1. State the outcomes of the research without interpretations in at least one paragraph of text. 2. Refer to statistical significance only if statistical tests (t-tests, ANOVA, etc.) were conducted. 3. Place statistical values (e.g. p-values) in parentheses after stating a result was significant. 4. Refer to tables and figures in parentheses at the end of sentences that reference these results. Discussion 1. State if your alternative hypothesis was supported or not supported o accept or reject the null hypotheses o usually incorporated into a paragraph for each set of hypothesis (if you tested more than one set) with a restatement of significant results and a discussion of prior research. 2. Refer to prior research with proper citations o How do your results compare to past research? o Use in-text citations for literature (see below) 3. Issues with the research methods o Explain what may have biased results o Give ways these problems could be reduced if the experiment were conducted again 4. Significance of the research o What new information has been gained by this project? o What overall conclusions can be made? (put the work into a broader context) o What implications does this research have for the field (e.g. management of resources, new laws that should be introduced, recommendations to policy makers or stakeholders)? o What are some follow-up questions arising from this work? o How might you investigate these follow-up questions?

102

GENERAL BIOLOGY LAB MANUAL – APPENDICES Tables 1. Tables need to be easy to understand (convey data as simply as possible) 2. Do not present raw data (i.e. data as you collected it) – instead, present summarized data (e.g. means with standard error) 3. Be sure to include units 4. Tables require captions (above the table) that describe the data contained within. o e.g. Table 1. The mean diameter (cm) of pine trees along a mountain slope. 5. Gridlines are not usually included in tables 6. Make a single table for all related data o Do not separate data by treatment, location, etc. unless necessary Figures 1. Figures need to be easy to understand (convey data as simply and clearly as possible) 2. Do not present raw data (i.e. data as you collected it) – instead, present summarized data (e.g. means with standard error) 3. Be sure to include units 4. Figures include graphs, diagrams, flow charts, art work, photographs, etc. – essentially any graphic that is not a table. 5. Figures require figure legends (below the figure) that describe the data represented o e.g. Fig. 1. The mean diameter (cm) of pine trees at three slope locations. Acknowledgements Acknowledgements are used to give credit to individuals that helped with the research, but are not authors of the paper. This might include technicians, reviewers, those who provided advice, etc. Funding sources are recognized and any permits that were required to do the research are reported. Literature Cited 1. Sometime title References or Works Cited. 2. Lists in alphabetical order all sources cited in the text (check the reference list to the internal citations prior to printing/turning in your work) 3. Formatting is critical – use the format provided below. 4. Be consistent with formatting 5. Use initials for first and middle names (when given) of authors 6. References should always have hanging indents

103

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Citations The primary literature used in scientific writing are peer-reviewed journal articles. For your lab reports, you will be expected to find such articles to support statements you make in your introduction and discussion, to compare with your findings, and to suggest further work. You may also use books as references. With few exceptions, websites are not considered reputable sources for scientific articles, and should not be used in lab reports. For this reason we have not provided guidelines for citing internet sources. In-text citations in scientific literature consists of the last name(s) of the author(s), and the year of publication. Do not report page numbers. If citing multiple sources in-text to support a single statement, list references in order by date. Examples: • One author: (Jones 1999) • Two authors: (Jones & Smith 2008) • More than two authors: (Jones et al. 2011) – “et al.” means “and others” • Multiple sources: (Jones 1999, Jones & Smith 2008) Literature Cited sections should use the format shown below. List references alphabetically with hanging indents. If citing multiple works by the same author, list these in order by date. • Journal article example, single author: Alexander, R.D. 1974. The evolution of social behavior. Annual Review of Ecology and Systematics 5:325–383. • Journal article example, multiple authors: Hanggi, P., P. Talkner, and M. Borkovec. 1990. Reaction-rate theory: fifty years after Kramers. Reviews of Modern Physics 62:251–341. • Book example: Brooker, R., E. Widmaier, L. Graham, and P. Stiling. 2014. Biology, 3rd ed. McGraw-Hill, New York, New York. • Book chapter example: Sargent, J.R., and K.J. Whittle. 1981. Lipids and hydrocarbons in the marine food web. Pages 491-533 in Analysis of Marine Ecosystems, A. Longhurst (editor). Academic Press, London, UK.

104

GENERAL BIOLOGY LAB MANUAL – APPENDICES

EXAMPLE LAB REPORT Chris North LIFE 1010 lab section 34 8 Nov 2015

A comparison of the rate of fermentation of corn and cane sugar by baker’s yeast (Saccharomyces cerevisiae)

Introduction Many organisms can carry out fermentation, a process that allows cells to produce adenosine triphosphate (ATP) even when they cannot perform cellular respiration due to lack of a final electron receptor, such as oxygen (Freeman et al. 2014). Fermentation occurs in animal cells during anaerobic exercise and is performed by many single-celled organisms in anaerobic environments (Freeman et al. 2014). Like cellular respiration, fermentation produces carbon dioxide (CO2) as a byproduct during the catabolic breakdown of glucose to release energy (Freeman et al. 2014). Fermentation also produces other byproducts, such as lactic acid in humans and ethanol in some yeast (Freeman et al. 2014). One species of yeast that can produce ethanol is baker’s yeast (Saccharomyces cerevisiae) (Legras et al. 2007, Hageman & Piškur 2015). Baker’s yeast has been used by humans to produce ethanol for consumption for millennia (Legras et al. 2007). More recently, people have explored the possibility of using ethanol produced by fermentation as an alternative to fossil fuels. Such “biofuels” can be produced from a variety of different materials or substrates, including cane sugar and corn (Groom et al. 2008). 105

GENERAL BIOLOGY LAB MANUAL – APPENDICES Our question was: Does S. cerevisiae ferment corn and cane sugar at the same rate? To address this question, we measured the rate of CO2 production by yeast given either cane sugar or corn as a substrate. Our null hypothesis was that the substrate used will not affect the rate of CO2 production by fermentation. Our alternative hypothesis was that the substrate used will affect the rate of CO2 production by fermentation. We predicted that cane sugar would be fermented at a faster rate than corn.

Methods To estimate the rate of fermentation of cane sugar and corn by yeast, we measured CO2 production in fermentation apparatuses over 40 minutes. Fermentation apparatuses consisted of a 50 mL centrifuge tubes with a 20 mL syringe attached to the lid by a Luer lock. A yeast solution was prepared by adding 35 g of dry baker’s yeast and 1 g NaCl to 1 L of water at 40 °C and agitating for 5 minutes. To each tube, we added 15 mL of the yeast solution and 1 g of substrate. We then secured lids, with syringes fully depressed, to the tubes. Every 10 minutes for 40 minutes, we recorded the displacement of the syringe which indicated how much gas had been produced in the fermentation apparatus. For the cane sugar treatment group we used Sugar in the Raw ® and for the corn treatment group we used corn meal. In addition to our two treatment groups (cane sugar and corn), we also had a negative control group to which no substrate was added and a positive control group to which we added 1 g of glucose. We performed three replicates in each experimental group. For each replicate, we calculated the rate of CO2 production over the 40 minute experiment. Averages and standard deviations (SD) were calculated for CO2 produced and rate of CO2 production for each experimental group. The rate of CO2 production was compared between the two treatment groups by a t-test performed with Microsoft Excel. 106

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Results Over 40 minutes, the positive control group produced the most CO2 (20.3 ± 0.6 mL, n = 3), while the negative control group produced the least (0.2 ± 0.1 mL, n= 3) (Fig. 1). In the same amount of time, the corn treatment group produced 4.9 ± 0.3 mL CO2 (n = 3) while the cane sugar treatment group produced 13.1 ± 0.3 mL CO2 (n = 3)( Fig. 1). The rate of CO2 production in the cane sugar treatment group (0.33 ± 0.1 mL/min, n = 3) was significantly higher (t-test, p < 0.001) than the rate of CO2 production in the corn treatment group (0.12 ± 0.1 ml/min, n =3) (Fig. 2).

Fig. 1. CO2 production by yeast (Saccharomyces cerevisiae) in fermentation apparatuses with different substrates over 40 minutes. Each 50 mL apparatus received 15 mL of yeast solution and 1 g of substrate. The negative control group received no substrate, while the positive control group received glucose. Each point on the figure represents the average of three replicates.

107

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Fig. 2. The average rate of CO2 production by yeast (Saccharomyces cerevisiae) in fermentation apparatuses when given cane sugar or corn as a substrate (n = 3). Each 50 mL apparatus received 15 mL of yeast solution and 1 g of substrate. The rate of CO2 for each replicate was calculated over 40 minutes. Carbon dioxide was produced at a significantly higher rate when cane sugar was the substrate than when corn was used (t-test, p < 0.001).

Discussion We found that S. cerevisiae produced CO2 at a higher rate when given cane sugar as a substrate than when given corn (0.33 ± 0.1 mL/min versus 0.12 ± 0.1 ml/min, p < 0.001; Fig. 2). We also observed that the production of CO2 was higher in the cane sugar treatment group over the entire experimental period of 40 minutes (Fig. 1). These results support our alternative hypothesis that the substrate used affects the rate of fermentation by yeast and allow us to reject the null hypothesis that that the substrate used has no affect the rate of fermentation by yeast. Furthermore, our results support our prediction that cane sugar would be fermented more quickly than corn. Our findings

108

GENERAL BIOLOGY LAB MANUAL – APPENDICES agree with other work which found that sugarcane yields more ethanol per capita than corn or sugar beets due to its higher glucose concentration (Renouf et al. 2008). Several potential sources of error exist with this study. For one, we discovered that gas was leaking from some of our fermentation apparatuses because the seal between the lid of the centrifuge tube and the tube was not airtight. Although we were able to use Teflon tape to prevent most leaks, it is possible that some tubes were still leaky, which could have biased our results. If we were to perform this experiment again we would use tubes with O-rings. Another potential source of error is that we did not completely fill our tubes with yeast solution. As a result, oxygen in the headspace may have resulted in cellular respiration by the yeast, which would affect our measurements of fermentation since respiration also produces CO2. However, when glucose is available S. cerevisiae still performs fermentation in favor of respiration even under aerobic conditions (Hageman and Piškur 2015), so this may not be an issue. Other potential substrates might also be effective for production of ethanol for biofuel use. For example, sugar beets are used in the United Kingdom (Renouf et al. 2008) and other food crops might be good candidates. It would be interesting to perform this experiment with different substrates. Also, fermentation of corn may have been lower than fermentation of cane sugar because some carbohydrates in corn are stored as starch and not glucose – could adding amylase, an enzyme that converts starch to glucose, result in fermentation rates of corn comparable to those of cane sugar? Could other factors, such as heat or pH, be manipulated to increase the rate of fermentation, and improve the yield of ethanol when corn is used as a substrate? Further studies need to be performed to determine if corn is a viable source of biofuel.

109

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Literature Cited

Freeman, S., K. Quillin, and L. Allison. 2014. Biological Science, 5th ed. Benjamin Cummings, San Francisco, California. Groom, M.J., E.M. Gray, and P.A. Townsend. 2008. Biofuels and biodiversity: principles for creating better policies for biofuel production. Conservation Biology 22:602-609. Hageman, A., and J. Piškur. 2015. A study of the fundamental mechanism and the evolutionary driving forces behind aerobic fermentation in yeast. PLoS ONE 10: e0116942. Legras, J.L., D. Merdinoglu, J.M. Cornuet, and F. Karst. 2007. Bread, beer and wine: Saccharomyces cervisiae diversity reflects human history. Molecular Ecology 16:2019-2102. Renouf, M.A., M.K. Wegener, and L.K. Nielsen. 2008. An environmental life cycle assessment comparing Australian sugarcane with US corn and UK sugar beet as producers for fermentation. Biomass and Bioenergy 32:1144-1155.

110

GENERAL BIOLOGY LAB MANUAL – APPENDICES

LAB REPORT RUBRIC Student: ____________________________ Late Penalty (10% per day, up to 72 hours): _____%

Total Score: =

_____% _____ / 50

Title & Introduction (20%) ____ (2) Title is descriptive and represents experimental question and/or findings. ____ (4) Relevant background information is included ____ (4) Literature citations are included to support background information where appropriate. ____ (4) Purpose and experimental question are clearly stated. ____ (4) Null and alternative hypotheses are both clearly stated. ____ (2) Prediction of outcome is given. ____ (20) Total

Methods (20%) ____ (4) Methods are clear ____ (4) Methods are complete ____ (4) Methods provide enough detail that the study could be duplicated. ____ (4) Descriptions of controls, sample size, and replication are included. ____ (4) Written in past tense and in paragraph form. ____ (20) Total

Results (20%) ____ (6) All results are clearly described in writing. ____ (2) Any trends, outliers, or interesting relationships are identified but not interpreted. ____ (2) Figures and/or tables are referred to in the text with figure/table numbers. ____ (3) All figures and/or tables clearly and correctly represent data. ____ (3) Figure axes are correctly labeled and include proper units and/or columns and rows in tables are clearly labeled with proper units. ____ (4) Each figure and/or table has a descriptive caption (called a figure or table legend) that provides enough information for a reader to understand and interpret the figure or table without referring to the written report. In other words, figures and tables are standalone. ____ (20) Total

111

GENERAL BIOLOGY LAB MANUAL – APPENDICES

Discussion (20%) ____ (4) Results are summarized and interpreted in light of the original experimental question and background information. ____ (4) Hypotheses (both null and experimental) are accepted or rejected with explanation. ____ (4) Literature is cited (at least two sources) to help explain data or conclusions, for comparison, or to suggest At lemodifications. ____ (4) Potential sources of error or bias are identified and their impact on the results are discussed. ____ (4) Relevant additional research questions arising from results are posed and further studies suggested. ____ (20) Total

Writing & Citations (20%) ____ (8) Lab report is clear, concise, and easy to read with complete sentences and few spelling or grammatical errors. Paper has been proofread and edited. ____ (2) Lab report includes section titles (i.e., Introduction, Methods, etc.), and is double-spaced. ____ (2) Scientific species names are properly given (e.g. Homo sapiens), and any abbreviations used are appropriate and clearly defined ____ (2) At least 3 peer-review journal articles are cited. ____ (2) Each in-text citation is included in the literature cited section and vice versa. ____ (2) Correct format (as described below) is used for both in-text citations and literature cited section. ____ (2) No direct quotes are used. ____ (20) Total

General Comments:

112