Biology 112 Cell Biology for the Health Sciences

Transcription

1 Biology 112 Cell Biology for the Health Sciences Lab Manual Portland Community College Cascade Campus By Troy Jesse Shannon Ansley Lisa Bartee Nancy Briggs Kendra Cawley Wes Dubbs Lisa Brown-Istvan Linda Fergusson-Kolmes Sandy Neps

2 Table of Contents SAFETY GUIDELINES AND GENERAL DIRECTIONS FOR BIOLOGY LABORATORY CLASSES... 3 LAB 1: THE METRIC SYSTEM AND THE SCIENTIFIC METHOD... 5 LAB 2: MICROSCOPE SKILLS LAB 3: PH AND BUFFERS LAB 4: BIOLOGICAL MACROMOLECULES LAB 5: DIFFUSION AND OSMOSIS LAB 6: ENZYMES LAB 7: FERMENTATION LAB 8: THE STRUCTURE OF DNA AND DNA REPLICATION LAB 9A: CELL CYCLE AND MITOSIS LAB 9B: MEIOSIS LAB 10: GENETICS APPENDIX A: GUIDE TO WRITING A SCIENTIFIC PAPER APPENDIX B: HOW TO MAKE A SCIENTIFIC FIGURE APPENDIX C: MICROWORLDS PROJECT APPENDIX D: PERIODIC TABLE OF THE ELEMENTS

3 Safety Guidelines and General Directions for Biology Laboratory Classes 1. Familiarize yourself with exits and evacuation procedures, and the locations and uses of the fire extinguisher, eyewash station, clean up materials for chemical spills, broken glass container, fire blanket, safety kit and emergency shower. 2. For your safety, eating and drinking, including water, are prohibited in laboratories by OSHA (Occupational Safety and Health Administration) and PCC regulations. Chewing gum, using tobacco products of any kind, and/or applying cosmetics are also not allowed in laboratories. Please leave all food and drink items on the shelves outside the lab rooms. 3. Please store all personal materials (backpacks, coats, etc.) in the cubicles or shelves in the laboratory rooms. 4. For your safety, if you are pregnant or have any other medical conditions that might necessitate special precautions in laboratory, please inform your instructor immediately. If you know you have an allergy to latex, insect bites and/or any specific chemicals, please inform your instructor. 5. Closed-toe shoes and appropriate, protective clothing must be worn in the laboratory. Long hair and dangling jewelry can be dangerous in lab. 6. Wear disposable gloves when handling blood and/or other body fluids, such as saliva or urine, or when touching items or surfaces soiled with blood or other body fluids. Wash hands thoroughly, immediately after removing gloves. 7. Report all spills and accidents to your instructor immediately. You will be advised of the proper clean-up procedures. 8. Students must follow all instructor-specified safety procedures. Students who do not comply with these safety guidelines may be excluded from the laboratory. 3

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5 Scientific Method Lab 1: The Metric System and the Scientific Method Objectives: 1. Explain the process of the scientific method. 2. Demonstrate the ability to generate hypotheses and test them experimentally. 3. Work effectively in groups. 4. Identify the dependent, independent and controlled (standardized) variables in experiments. 5. Identify the control group in an experiment and know the difference between a control group and a variable. 6. Know and follow the safety rules of the biology laboratory. 7. Be able to use standard metric units for volume, mass and length. 8. Know the meaning of metric prefixes (centi, kilo etc.) and be able to use these units. 9. Be able to use scientific notation to express measurements. 10. Be able to multiply and divide numbers expressed in Scientific Notation. 11. Know the difference between accuracy and precision. 12. Be able to use the basic lab tools for measuring volume, mass and length. Vocabulary: hypothesis (plural, hypotheses), prediction, theory, variable, independent variable, dependent variable, experimental group, control group, controlled (standardized) variable, repetition, sampling error, metric, International System of Units, SI, liter, gram, meter, degrees Celsius ( C), Scientific Notation, kilo-, centi-, milli-, micro-, nano-, exponent, accuracy, precision, beaker, Erlenmeyer flask, beaker, graduated cylinder, pipettes, meniscus, pi-pump, taring. The Metric System and Basic Measurement Techniques Introduction: In our everyday lives, we use a system of measurement that uses units such as gallons, pounds and feet. However, many other countries and most importantly for this course, the scientific community, use the metric system (International System of Units; SI). It is important that you can use and understand these units, because they are the standard way that scientists and health care professionals of all kinds communicate with each other. Table 1 contains a comparison of some of the most common American (also called English or Imperial units) and SI units. 5

6 Scientific Method Table 1. Conversions between some American and Metric (SI) units. Volume Mass Length 1 gallon =3.79 liters (L) 1.06 quarts = 1 liter (L) 1 fluid ounce = 29.6 milliliters (ml) 2.20 pounds = 1 kilogram (kg) 1 pound = kilograms (kg) 1 ounce = 28.3 grams (g) 1 mile = 1.61 kilometers (km) 1.09 yard = 1 meter (m) 1 inch = 2.54 centimeters (cm) The base units that you should be familiar with are the liter (L) for volume, the gram (g) for mass and the meter (m) for length. It is not the focus of this lab, but it is also useful to know that the unit for temperature is degrees Celsius ( C). These basic units are organized in factors of 10. The prefix added to the basic unit indicates how much smaller or how much larger it is than the base. The prefixes have the same meaning whether they are added to units of volume, mass or length. For example, kilograms, kiloliters and kilometers are units that are all 1000 times greater than grams, liters and meters. If you use a computer a great deal, you will be familiar with a unit of memory called a kb or a kilobyte. The same prefix rules apply; there are 1000 bytes in a kilobyte. So, once you learn the meaning of the prefixes, you are set no matter what you are measuring! Table 2 contains a list of metric prefixes. Prefix Table 2. Metric prefixes. Size relative to base unit Size relative to base unit expressed as an exponent of 10 giga billion 10 9 mega million 10 6 kilo thousand 10 3 centi hundredth* 10-2 milli thousandth* 10-3 micro millionth* 10-6 nano billionth* 10-9 Note - This table represents each prefix as a fraction of the base unit. For example, one centimeter is 1/100, or a hundredth, or 10-2 of a meter. Sometimes when working with prefixes that indicate a unit SMALLER than the base (i.e. centi-), it is sometimes easier to think about how many of the small units are in the larger base unit for example, 1 meter contains 100 centimeters (10 2 cm). 6

7 Scientific Method Scientists sometimes work with very large or very small numbers. To avoid having to write many beginning or trailing zeros, scientists use a type of shorthand for numbers called Scientific Notation. When you use Scientific Notation, each number is expressed as a decimal multiplied by a power of ten. Numbers greater than 1 would have a positive exponent to represent the power of 10, and numbers less than 1 would have a negative exponent to represent the power of 10. The power of 10 is indicated by the exponent (Table 3). For example, the number 353 would be expressed 3.53 x 10 2, and the number would be expressed as 3.53 x Practice converting numbers to and from scientific notation by filling in the blank spaces in Table 4 Table 3. Exponents and powers of /10, / / / x x 10 x , x 10 x 10 x Table 4. Expressing numbers in Scientific Notation. Number in Standard Form Number in Scientific Notation x x x x

8 Scientific Method Multiplying Numbers Expressed in Scientific Notation When you multiply two numbers expressed in Scientific Notation, you multiply the decimal portion of both numbers and then add the exponents. e.g. (2.5 x 10 3 )(2.0 x 10 2 )= (5.0 x 10 5 ) or (3.0 x 10-2 )(2.0 x 10-4 )= (6.0 x 10-6 ) or (4.0 x 10 4 )(2.0 x 10-2 )= (8.0 x10 2 ) Dividing Numbers Expressed in Scientific Notation When you divide two numbers expressed in Scientific Notation, you divide the decimal portion of both numbers and then subtract the exponents. e.g. (4.0 x 10 5 ) = (2.0 x 10 3 ) (2.0 x 10 2 ) or (5.0 x 10-2 ) = (2.0 x 10 2 ) (2.5 x 10-4 ) or (4.0 x 10 4 ) = (2.0x10 6 ) (2.0 x 10-2 ) Accuracy and Precision To interpret a measurement of any kind, it is important for a scientist to have an idea of both the accuracy and the precision of the measurement. Accuracy refers to how close the measurement is to the true value. How many of you have stood on a bathroom scale that wasn't working properly? The scale may have given you a measurement of your weight, but if it wasn't close to the real value, it wasn't very accurate. However, if you climbed back on that scale 10 times in disbelief, you might always get the same value. It wouldn't be accurate, but it would be precise. Precision refers to how consistently a measurement can be reproduced. It is important in a laboratory setting to choose the right tool. For example, you would not use a 250 ml beaker to try to measure 1 ml, or a bathroom scale to try and measure something with a mass of 10 g. It is also important to know how to use each tool properly to make sure measurements made in lab are as accurate and precise as possible. 8

9 Scientific Method Introduction to the Basic Lab Tools for Measuring Volume, Mass, and Length Erlenmeyer flask beaker graduated cylinder pipette Volume Figure 1. Measuring tools for volume. The basic unit of measurement for volume is the liter (L). 1 L =10 3 ml=10 6 µl equipment drawer should contain a variety of measuring tools. Erlenmeyer flasks and beakers may have volume markings, but they are approximate and should not be used when you need a measurement that is accurate. Graduated cylinders and pipettes of different sizes are much more accurate and precise if used properly. Liter (L) Milk would be sold in liters. One liter of milk would be just a little bit more than a quart. Milliliter (ml) 1 L = 1000 ml (10 3 ml) A tablespoon is about 15 ml. The plastic measuring cups that come with some cough syrups often have ml marks on the side. Microliters (µl) 1 L =1,000,000 µl (10 6 µl); 1 ml = 1,000 µl (10 3 µl) You would use a micropipette to measure microliters. This is a very small unit of volume. 9

10 Scientific Method It is important when measuring a liquid to be aware that how you look at the glassware can affect your measurement a great deal. When you are using glassware to measure a volume of a solution, always: meniscus 1. View the volume markings on the glassware at eye level. 2. Read the volume from the bottom of the meniscus. A meniscus forms when the liquid in the container 'creeps' up the sides a little bit. Exercise 1: Measuring With a Pipette Equipment Needed: 2 beakers tap water 1 ml pipette pi-pump for 1 ml pipette 5 ml pipette pi-pump for 5 ml pipette Procedure: 1. Gather all equipment needed, and read through this procedure before beginning. 2. Take a close look at the markings on both the 1 ml and the 5 ml pipettes. There are many sizes of pipettes. The same size pipette may also vary in the way it is marked. The top of the pipette will indicate the largest and smallest volumes appropriate to measure with that pipette. For example, it may say 1 ml in 1/100. This means the pipette measures 1 ml from the 0 line to the 1 ml line. The smallest distance between lines on the pipette will then represent 1/100 of a ml (0.01 ml). Your instructor may add additional details depending on the nature of the equipment in your lab. Record those details for reference in the space below. 3. To draw liquid into the pipette various kinds of pumps may be used. Many years ago, scientists used to use their mouths to suck up the liquid. Needless to say, you should NEVER mouth-pipette. Your lab will be equipped with a pipette pump (abbreviated pi-pump). Your instructor may demonstrate the use of the device found in your lab. 4. Fill one of your beakers approximately half-full with tap water. 5. Attach the appropriate pi-pump to the top of the 1 ml pipette. 10

11 Scientific Method 6. Put the tip of the pipette into the solution. Make sure that you always immerse the tip of the pipette completely or you will get bubbles. Make sure that you hold the pipette as vertical as possible. Draw up 1 ml of water into the pipette. Make sure that you are reading the markings from eye level and from the bottom of the meniscus. Dispense the water into the empty beaker. Repeat until you are comfortable handling the equipment. Never hold the pipette upside down once it has been used. Be aware of where you put the end of a used pipette. 7. Repeat steps 5-6 with a 5 ml pipette. 8. Discard the pipettes as directed by your instructor. This is important because you do not want to create a safety hazard; also, some pipettes are recyclable. Mass The basic unit of measurement for mass is the gram. 1g =10 3 mg=10 6 µg=10 9 ng The most commonly encountered units are the kilogram (kg), gram (g), milligram (mg), and microgram (µg). Kilogram (kg) 1 kg= 1000 g (10 3 g) You would communicate your body weight in kilograms. Flour and sugar would be sold in kg. A 5 kg sack of oranges would be about 11 pounds. Gram (g) You would buy spices by the gram. A US nickel weighs about 5 grams when new. Milligram (mg) 1 g = 1000 mg (10 3 mg) The amount of active ingredient in an over the counter pain reliever would be expressed in milligrams. For example, each Extra Strength Tylenol tablet contains half a gram, or 500 milligrams, of acetaminophen. Micrograms (µg) 1 g =1,000,000 µg (10 6 µg); 1mg =1000 µg (10 3 µg) A small grain of sand would have a mass of about 3 µg. Nanogram (ng) 1g =1,000,000,000 ng (10 9 ng); 1 mg =1,000,000 ng (10 6 ng); 1 µg = 1,000 ng (10 3 ng) The average human cell has a mass of 1 ng. We don't have balances that can even come close to measuring anything in this range. This is tiny! 11

12 Scientific Method Exercise 2: Using a Top-Loading Electronic Balance Equipment Needed: Top-loading electronic balance Weigh boat Small items such as beans, rocks or pennies Procedure: 1. Gather or locate all equipment needed, and read through this procedure before beginning. 2. Find the electronic top loading balance provided for your group. Make sure it is plugged in and turned on. Make sure that it is set to the correct mode for weighing in grams. Note whether the model you have will determine the mass to 1 decimal place or two. 3. Place the weigh boat on the balance pan. Use the zero feature to tare the balance. Taring, or zeroing, the balance resets the readout artificially to zero, even though the weigh boat has its own mass. This is a convenient feature, because now the read-out will give you directly the mass of anything you add to the top. 4. Place 5-6 beans in the weigh boat and note the mass. Length The basic unit of measurement for length is the meter. 1 m= 10 2 cm=10 3 mm=10 6 µm=10 9 nm The most commonly encountered units are the kilometer (km), meter (m), centimeter (cm), millimeter (mm), and micrometer (µm). You will work with millimeters and micrometers in the microscope lab. This next exercise will focus on making sure that you are familiar with a meter stick, and the small metric rulers found in your lab equipment drawer. Here are some examples of what units are used, using something that might be familiar to you. Kilometer (km) 1 km =1000 m (10 3 m) Speed limits in Canada and Europe would be in km/hr. A 5k race is about 3 miles. Meter (m) A meter is just a little bit longer than a yard. In a metric country, you would buy fabric in meters. You might run a 100 m dash instead of a 100 yd dash. Centimeter (cm) 1 m = 100 cm (10 2 cm) You would use centimeters in places where you are used to using inches. 12

13 Scientific Method Millimeter (mm) 1m = 1,000 mm (10 3 mm); 1 cm = 10 mm (10 1 mm) You can still see things that are 1-2 mm wide but they are relatively small. The average quarter is a little over a millimeter thick. Micrometer (µm) 1 m = 1,000,000 µm (10 6 µm); 1 cm =10,000 µm (10 4 µm); 1 mm = 1000 µm (10 3 µm) This is the unit of measurement when you are looking at a cell or components of a cell. If something is about 100 µm, you will still be able to see it with a light microscope. The diameter of human hair ranges from 17 µm to 181 µm. Nanometer (nm) 1 m = 1,000,000,000 nm (10 9 nm); 1 cm = 10,000,000 nm (10 7 nm); 1 mm =1,000,000 nm (10 6 nm); 1 µm = 1,000 nm (10 3 nm) This is a measurement for things like individual membranes in cells. The plasma membrane that encloses a single cell is 5 to 10 nm wide. You would need an electron microscope to see something that was only a few nanometers wide. Exercise 3: Working with Meter Sticks and Metric Rulers Equipment Needed: Meter stick Small metric ruler with cm and mm markings Procedure: 1. Gather or locate all equipment needed, and read through this procedure before beginning. 2. Locate a meter stick. Look at the markings. How many centimeters are in a meter? 3. Using either the meter stick (or a measuring tape, if one is taped to the wall in your lab), and with the help of a partner, measure your height in meters: 4. Find something in the lab that is about a meter in length. 5. Locate a small metric ruler. Find the centimeter markings. Each centimeter should be divided up into 10 mm. Measure 2-3 items in the lab in centimeters. Find the millimeter markings. Repeat the measurement in millimeters (mm). 13

14 Scientific Method Converting Between Units Most of you will already have figured out that you didn't really have to measure something in centimeters and then measure it again in millimeters. All you have to do is be able to convert between units. It may seem simple but the ability to convert between units of measurement is crucial. A number of years ago NASA's Mars Climate Orbiter was lost in space because engineers did not convert from the American units to metric. This error sent the craft too close to the surface of Mars. $125 million was lost over a simple conversion (CNN.com, 1999). Recent studies indicate that giving the wrong dose, often because of a conversion error, is the second most common mistake made by student nurses (Wolf et al., 2006). A seemingly small mistake can have tragic consequences. A fifteen-day-old British baby died in 2002 when the nurses determining the appropriate dosage of his medication made a conversion error, slipped a decimal, and administered 220 µg instead of 22 µg (BBC News, 2005). Example 1: A BI 112 student is 1.75 m tall. How many centimeters is that? Use the prefixes in Table 2 to determine the conversion factor. 1 cm = 10-2 m or a hundredth of a meter. So in 1 meter you would have 10 2 cm (100 cm) Example 2: The average capillary diameter is about 6.2 µm. How many cm is that? Literature Cited: BBC News. 2005, May 5. Baby died after decimal error. < Accessed September 10, CNN.com. 1999, September 30. NASA s metric confusion caused Mars orbiter loss. < Accessed September 10, Wolf, Z., R. Hicks, and J. Serembus Characteristics of medication errors made by students during the administration phase: a descriptive study. Journal of Professional Nursing 22:

15 Scientific Method The Scientific Method Introduction: The Scientific Method involves the systematic accumulation of knowledge that is based on things that can be directly or indirectly experienced. So, science is an organized body of facts information derived from observation, experimentation, and evaluation and it is also a method for solving problems. Over the years, human societies have accumulated vast amounts of information. Science is the systematic study of the world and its components. There is nothing mysterious about scientific reasoning. In fact, most people use a scientific approach every day in solving problems and making decisions. You do not need special training to decide whether conclusions are justified from the data given. You must simply follow the rules of logic. The basic features of the scientific method are: 15 Observations or Measurements Questions Hypothesis: tentative explanation of how something works Predictions: what should happen in a given situation if your hypothesis is correct Experiments: alter only one variable at a time; for that variable, provide a control group Substantiated Hypothesis New/Revised Hypothesis New Experiment Example: You get into your car and turn on the ignition switch with your car key and nothing happens. This observation leads to a hypothesis that the car failed to start because the battery is dead. From this hypothesis you predict that if the battery is dead, then you should not be able to turn on the lights. You then perform an experiment to test your hypothesis by trying to turn on the lights while the key is in the on position in the ignition. If the lights work, you discard the original hypothesis and try another one. If the lights failed to come on, you have supported your hypothesis. You have not proven your hypothesis because there may be another reason the lights have failed to work properly. Now, assume the lights in your car worked just fine when you tried the switch. You discard the dead battery hypothesis and come up with a new one that the car will not start because of a loose connection to the starter. To test this revised hypothesis, you open the hood and check the connections to the starter motor. Sure enough, the wire is loose. This supports your revised hypothesis, but the best way to test the hypothesis is to tighten the wire and try to start your car. If it starts, you have supported your revised hypothesis. This simple process is all there is to the scientific method. The catch is that it must be rigorously applied without short cuts. A valid hypothesis must suggest a cause and effect relationship and involve only one cause at a time. In addition, it must be testable and falsifiable. Good hypotheses lead to logical

16 Scientific Method predictions. Predictions are generally in the form of an if-then statement. The if clause simply states the hypothesis, and the then clause: 1. Suggests altering one causative factor in the hypothesis. 2. Predicts the outcome of the experiment. Once you have a hypothesis and prediction, you still have to design a proper experiment. This is generally the hard part of the scientific method. Good experiments should be designed to test only the factor (variable) suggested as the cause in the prediction. The best way to do this is to use two groups of subjects that are treated identically except with respect to the one factor under study. One group the experimental group has the factor (variable) under study altered, while the other group the control group does not. The factor under study is the independent variable. If you hypothesize that a factor makes a difference, you will need to have some way to compare your experimental group with the control group. Often you will try to measure or quantify a change in some way. The factor that is measured or counted is the dependent variable. Factors that you try and keep the same between experimental groups are controlled or standardized variables. Notice that controlled variables are not the same thing as a control group. Scientific knowledge is more than just collections of unconnected facts. Rather, it consists of numerous theories. Theories are underlying principles of science that are substantiated by many different lines of evidence and have never been proven false through repeated sets of prediction and experimentation. Unfortunately, the word theory is often misused in everyday language to mean hypothesis. Evolution of species is a theory; that flight feathers in birds evolved from feather-like structures that provided warmth in proto-avians is a hypothesis. It is impossible to test a theory with only one experiment. A theory is respected and generally considered to be true if it has been continually tested, but never falsified. This does not mean the theory is correct. It may be discarded as new scientific evidence is gathered. More often it will be replaced or modified. This requires that scientists remain open-minded about theories and be willing to look hard at any new evidence that questions the validity of the current theory. In order to remain open-minded, scientists try to develop critical thinking skills. Critical thinking in the context of the scientific method is the ability to distinguish between beliefs (not necessarily based on evidence) and knowledge (facts that are well supported by evidence). These skills provide us with a means of analyzing problems, issues, and information, objectively. The primary requirements are an open mind and an active approach to acquiring information. Do not accept everything you hear or read (even if done scientifically). You must define the issue or problem, and then examine the information and evidence available for both sides of the issue. These skills are summarized below. 16

17 Scientific Method Applying Critical Thinking Skills to the Scientific Method 1. Understand and define all terms. 2. Question the methods by which the facts were derived. Were facts derived from experiments or direct observation? If by experiment, were experiments well-executed? Did the experiments include a control group? Were there a sufficient number of subjects in the experiment? Has the experiment been duplicated by other scientists? Did they obtain the same results and conclusions? Were appropriate variables controlled (standardized)? 3. Question the source of the information. Is the source reliable? Is the source an expert? 4. Question the conclusions. Are the conclusions appropriate? Was there enough information to make the conclusions? 5. Uncover the assumptions and biases. Was the experimental design biased? Do underlying assumptions affect the conclusions? 6. Tolerate ambiguity. Do not expect all of the facts to be established by a single study. Expect controversy. 7. Examine the big picture. Look for multiple causes or effects. Look for hidden effects or relationships. 17

18 Scientific Method Exercise 4: Black Box Experiment You will work in groups of three or four; each group will receive a closed box containing unknown items. Your assignment is to determine the contents of your box by designing experiments that test hypotheses. Remember to have controls for your experiments. Groups will be required to present their final hypothesis as to what is in their box at the end of class, at which point check with your instructor to see how close you are (NO PEEKING). Your instructor may have you hand in the data sheet(s) at the end of lab. The following items may be in your box; there are no mystery objects: Bush beans paper clips peanuts Styrofoam quads plastic beads cotton balls Hint: There are numbers written on the bottom of your box: for example, That means that there are 7 of one type of item, 14 of another type, and 6 of a third type. This information, in combination with the availability of balances and test boxes/items should be useful to you. Good luck! Example: 1. Observations: Shake, listen, feel, etc. 2. Question: What is in the box? Hypothesis including prediction: There are marbles in the box. If there are marbles in the box...then I will hear them roll when I tilt the box gently. Experiment: Put some marbles in a test box. Tilt both your experimental box and your test box and listen. Think about how to construct this experiment to avoid bias Experimental Data: Both boxes are tilted gently. Rolling sounds are heard in the control but not in the experimental box. Conclusion: The hypothesis is disproved; there are no marbles in the box. The process for determining what you think the contents of your box are will be the result of many different item-by-item hypotheses and resulting experiments. Pick three of your hypotheses and resulting experiments and record them on the included sheet (p.25). Your instructor may choose to have your group present your theory (based on a number of different hypotheses that you have tested) to the class and then reveal the contents of the black box! 18

19 Scientific Method Things to Think About: 1. How are you going to test your hypothesis or hypotheses? What will your controls be? What is your experimental group? What underlying assumptions will affect your conclusions? (Note: some of your answers may be modified based on experience once we begin the lab.) 2. Sometimes scientific data is ambiguous. Think about ambiguity in the Black Box (there is not just one right answer; be prepared in lab to be thinking about what data may be ambiguous). 3. Even if you have clearly ruled out one hypothesis, and supported the other, you should hesitate to accept the final conclusions based on a single experiment. Repetition obtaining the same results after several different experiments helps to solidify the correctness of the hypothesis. Repetition also helps to avoid problems of sampling error. Sampling errors occur when you collect data from only part of a population (e.g. 10 birds or 100,000 people) because you only have an estimate of the true value of whatever you are measuring. To get the true value, you would have to collect data from every member of the population, and this is usually not practical. In general, the larger the sample size the more accurate the estimate. It also matters how much members of the population vary from each other. In the Black Box lab, think about the variation in the population of peanuts (e.g. for mass or length) compared to the variation in the population of paper clips. You might expect the average mass of 10 paper clips to be a much better estimate than the average mass of 10 peanuts. Variation in populations must be acknowledged and accounted for; it can be assessed by applying certain statistical procedures to your data. Think about how this affects research on human health! For example, the Nurses Health Study began in 1976 with 121,700 women. It was scheduled for completion in August of 2007 (NHS, 2007). The number of participants has fluctuated over the years, but it has produced landmark information about the relationships between lifestyle and health because of the large sample size. Literature Cited NHS The Nurses Health Study. Accessed Sept 13,

20 Scientific Method Black Box Experiment Data Sheet Names of Students in Group: Experiment 1 Observations Question Hypothesis and Prediction: Experiment and Procedure Independent Variable Dependent Variable Controls Constants Results: Conclusion: 20

21 Scientific Method Black Box Experiment Data Sheet Names of Students in Group: Experiment 2 Observations Question Hypothesis and Prediction: Experiment and Procedure Independent Variable Dependent Variable Controls Constants Results: Conclusion: 21

22 Scientific Method Black Box Experiment Data Sheet Names of Students in Group: Experiment 3 Observations Question Hypothesis and Prediction: Experiment and Procedure Independent Variable Dependent Variable Controls Constants Results: Conclusion: 22

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24 Scientific Method Scientific Method, Metric System and Safety Study Guide Questions Be able to answer for a quiz: 1. What are the standard metric units for volume, mass and length? 2. What is the standard metric unit for temperature? 3. What does the prefix kilo- tell you about the relationship of a unit to the base unit (e.g. kilogram to gram)? Be able to answer the same question for centi, milli, micro and nano. 4. How would you represent a number such as 3,456 in Scientific Notation? 5. How would you represent a number such as in Scientific Notation? 6. How would you multiply numbers written in Scientific Notation? 7. How would you divide numbers written in Scientific Notation? 8. Write the number 10,000 as a power of 10 with the correct exponent. 9. Which of the following tools would be most appropriate for measuring 1 ml of water: 500 ml beaker, 250 ml Erlenmeyer flask, 500 ml graduated cylinder, 5 ml pipette or 1mL pipette. Be able to explain your answer in terms of accuracy and precision. 10. Which top loading balance would be more accurate; one that recorded mass to one decimal place or one that recorded mass to two decimal places? Would this affect the precision? 11. What is the purpose of a weigh boat or weigh paper? 12. What is the purpose of being able to tare or zero a balance? 13. Understand the difference between a hypothesis and theory. 14. Understand the importance of a prediction in a well-crafted hypothesis. 15. Be able to identify the independent, dependent, and controlled variables in an experiment. For example, try identifying the independent and the dependent and the controlled variables in the experiment described below: Dr. X is interested in the effect of weight training on the density of mitochondria in the skeletal muscles of athletes. She chooses 18 year old, male swimmers as her test population. She has measured the mitochondrial density in one of the calf muscles of each swimmer before a regimen of weight training and then again after weight training. She has 40 swimmers participate in the study. Data is collected from each one. 16. Be able to identify the control group and the experimental group in an experiment, if appropriate. 17. Be able to locate important safety features in the lab. 18. Be able to contact the appropriate agency in case of emergency. 19. Know the location of all emergency exits and emergency equipment appropriate to your lab room (use the safety handout as a guideline for what you need to know). 24

25 Scientific Method Practice Problems for Quiz Volume 1) 5 L = ml 4) 5000 ml= L 2) 100 ml = µl 5) 10,000 µl = ml 3) 1600 ml = L 6) 4000 µl = ml Mass 1) 5 kg = g 4) 5000 ng = µg 2) 100 mg = µg 5) 10,000 µg = mg 3) 1500 g = kg 6) 4000 mg = g Length 1) 5 m = cm 4) 5000 m = km 2) 100 mm = cm 5) 10,000 nm = µm 3) 67 cm = m 6) 4000 µm = mm 25

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27 Microscope Skills 27 Lab 2: Microscope Skills Objectives: 1. Be able to effectively use a compound microscope. a. Learn structures and functions of a compound microscope. b. Correctly use a compound microscope. c. Correctly put a compound microscope away. d. Know what is meant by the following terms (be able to calculate if appropriate): resolution/resolving power, depth of field, field of view, working distance, parfocal, total magnification. 2. Be able to estimate the size of a specimen in millimeters (mm) and micrometers (µm). 3. Be able to make a wet mount slide. Vocabulary: compound microscope, resolve, resolving power, oil immersion, ocular lens, eyepiece, binocular tube, stand, revolving nosepiece, objective lens, total magnification, power switch, light intensity, longitudinal adjustment knob, transverse adjustment knob, coarse adjustment knob, fine adjustment knob, lamp, base, iris diaphragm lever, filter holder, condenser, stage, specimen holder, diopter adjustment ring, pointer, pre-focusing lever, condenser height adjustment knob, field of view, parfocal, working distance, stage micrometer, wet mount Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: The microscope is an important tool for understanding the world around us. The limit to the resolution of the average human eye is about 0.15 mm. It is not possible for most people to resolve (or make out the details on) anything smaller than that. However, most plant and animal cells are smaller than 0.15 mm. The ability to view and study things that are below the resolution of the human eye is a critical tool for scientists and health care professionals. The ability to use a microscope with confidence is an important skill for all health sciences students. There are many different kinds of microscopes. The microscope you will work with in today's lab is a compound microscope. When you see an image through a compound microscope, the light has passed through two different magnifying lenses before it gets to your eye. Thus, the magnification is compounded or multiplied. The microscopes in lab will magnify a specimen up to 1000 times (we call that 1000x)! This will allow you to get a good look at even something as small as a bacterium. There is a limit to how much a compound light microscope can magnify an image so that more details can be seen or resolved. Resolving power is a measure of the ability to distinguish two objects as separate. The physics of visible light limit the resolving power of a light microscope to about 0.2 µm. Electron microscopes are able to form images of structures that are smaller than 0.2 µm because beams of electrons are used instead of light, so the resolving power is higher. Electron microscopes are rather large and very expensive, but the images are wonderful in their detail. Transmission electron micrographs are made by directing

28 Microscope Skills the electrons through thin sections of the sample. They show highly detailed images based on the degree to which electrons can move through the substance. Traditional scanning electron micrographs show surface details: the object is coated with an electron-dense material (usually gold) and then bombarded with electron beams, which bounce off in much the same way that visible light bounces off objects we can see, revealing their three dimensional nature. When you look at pictures of cells and cell structures, consider whether the image is more likely to be from an electron microscope or a light microscope. Your sense of this will improve as you look at more images. Within the limits of the resolving power of visible light there are many techniques that make light microscopy more powerful. Staining is one way of enhancing an image. If you make a wet mount of cheek cells from your own mouth, you will find that they are quite difficult to see. Addition of a small amount of methylene blue stain makes finding them, and seeing detail within them, much easier. Most of the prepared slides you will look at this term have been stained in some way, and you will also use stains on your own wet mounts to help you see things more clearly. Another technique that is used to enhance resolution is oil immersion. Light bends when it passes from one material to another. Some of the light traveling from the specimen being viewed on its way to the microscope to make an image you can see, will bend and escape when it passes through the air space between the slide and the microscope. Light bends less when it passes through oil compared to air, so putting oil in between the microscope lens and the specimen will let more of the light traveling from the specimen on the slide actually get to the microscope. Some microscopes have special lenses that are designed to work best with oil as a medium for the light path. You will learn how to use these lenses in lab today. A good-quality student laboratory microscope is a surprisingly expensive and delicate piece of equipment. Misuse can damage a good microscope very quickly. It is very important to learn how to use, clean and store a compound microscope properly. Please pay close attention to the directions in this lab manual and to those given by your instructor. As you work through the following exercises, make sure you completely answer the questions within each exercise. 28

29 Microscope Skills Exercise 1: Structures on a Compound Microscope and Their Function Equipment Needed: 1 compound light microscope per person Procedure: Locate all of the following parts of your microscope (and on Figure 1). 1. Ocular Lens (Eyepiece): magnification value located on upper surface or outer rim. Caution: Do not remove oculars!! Dirt or dust may enter the system. 2. Binocular Tube: two ocular lenses and tubes = binocular head (microscopes with one ocular are monocular; three oculars are trinocular). 3. Arm: carry with one hand around this part (other hand under base). 4. Revolving Nosepiece: rotates to move the objective into place. Use ring on nosepiece to move the objective lenses DO NOT grab objective lenses to rotate nosepiece. 5. Objective Lens: the magnification value is located around the edge. Total magnification = objective value multiplied by ocular value. Note: objectives with a power 90x or above are designed to be used ONLY with immersion oil and MUST be cleaned thoroughly after using oil. 6. Power Switch: turns light source on/off. O is off and is on. Turn microscope off if leaving it for more than a few minutes. 7. Light Intensity Knob: controls the intensity of the light passing through the slide. Turn to the lowest setting before turning the microscope on/off. 8. Longitudinal Adjustment Knob: controls slide movement from front to back. 9. Transverse Adjustment Knob: controls slide movement from side to side. Do NOT move slide with your fingers! This may damage the mechanism that allows the specimen holder (#18) to move the slide. 10. Coarse Adjustment Knob: used BEFORE fine adjustment to initially focus the specimen. Turning the knob causes the stage to move up or down. 11. Fine Adjustment Knob: fine focuses the image. Turning the knob causes the stage to move up or down, but the movement is very small. Use this knob for any objective other than the scanning (4X) objective. 29

30 Microscope Skills 12. Lamp: provides a light source. 13. Base: carry with other hand under base (other around microscope stand). 14. Iris Diaphragm Lever: controls amount of light transmitted by opening/closing. 15. Filter Holder: may hold a light filter. You will not have to change the filter for this lab but it is possible to do so for more advanced microscopy techniques. 16. Condenser lens: focuses and concentrates light on the specimen. 17. Stage: flat structure on which the microscope slide is placed. 18. Specimen Holder: mechanism that holds the slide in place. Pull back the hook on the right, insert a slide, and slowly release the hook so it does not crack the slide. 19. Diopter Adjustment Ring: compensates for differences in sight between right and left eyes. 20. Pointer: right ocular may have a pointer this allows you to move a slide to line up a specimen or structure with the pointer. 21. Pre-focusing Lever: in locked position, prevents stage from being moved up by the coarse adjustment to protect the objective (and microscope slide). When finished using the microscope, move to the unlocked position. Check whether locked is the up-or-down position; it varies by model. NOTE: Not all microscopes have this feature. 22. Condenser Height Adjustment Knob: moves condenser (and iris diaphragm) up or down. For most microscopic work, position condenser at its highest position. DO NOT confuse the set screw that holds the condenser lens and iris diaphragm in place with the condenser height adjustment knob. The set screw is silver, the adjustment knob is black and is found behind the condenser lens assembly. 30

31 Microscope Skills 19: Diopter adjustment ring 2: Binocular tube 4: Revolving nosepiece 3: Arm 1: Ocular lens 20: Pointer 5: Objective lenses 21: Pre-focusing lever 10: Coarse adjustment 11: Fine adjustment 18: Specimen holder 17: Stage 16: Condenser & filter holder 22: Condenser height adjustment 8: Longitudinal adjustment 14: Iris diaphragm lever 6: Power switch 7: Light intensity knob 12: Lamp 13: Base 9: Transverse adjustment Figure 1: Compound microscope parts Assigned Microscope #: Does your microscope have a pointer? Yes/No 31

32 Microscope Skills Exercise 2: Proper Use of a Compound Microscope Equipment Needed: Prepared slide of letter "e" Prepared slide of crossed threads Procedure: 1. Lift / carry / move microscopes with two hands one under the base and the other around the upper arm of the stand. 2. Do not slide a microscope to move it on a table it may damage parts, including the light bulb. 3. Carefully remove the cover (if present), taking care not to catch / tear it on microscope parts. 4. Make sure that the light intensity knob is at its lowest setting before plugging in the microscope. 5. Make sure that the stage is fully lowered by using the coarse focus knob. 6. Make sure that the lowest power objective (4X) is clicked into position. Note: The magnification on the lowest power objective is 4X, indicating that the image is magnified 4 times compared with the naked eye. On a compound microscope, additional magnification is offered by the eyepiece. On a microscope with a 10X eyepiece, the total magnification is 4 x 10, or 40X (the image appears 40 times larger than it would to the naked eye). When specifying magnification, always use the total magnification. Total magnification What is the power of the ocular lens on your microscope? What would the total magnification of a specimen be if you were using: 4X objective lens. Total magnification of image 10X objective lens. Total magnification of image 40X objective lens. Total magnification of image 100X objective lens. Total magnification of image 7. Make sure that the pre-focusing lever (if present) is in the unlocked position. 8. Slowly move the light intensity knob to a higher setting (if no light appears in the filter mount area, return light setting to the lowest, click the power switch, and repeat). 32

33 Letter e BI 112 Microscope Skills Exercise 2a Exploring the Field of View (letter e slide) 9. Locate the prepared slide with the typed letter "e". Carefully position the slide for viewing. If the microscope you are using has arms that hold the slide, then gently retract the arms of the specimen holder and place the slide on the stage between the arms. Do not attempt to get the arms of the specimen holder over the slide. Do not allow the holder to spring back against the slide; this can cause damage to the slide. 10. Orient your slide so that the letter e is right-side up. Center the letter "e" area on the microscope stage directly under the objective lens. In this case the letter "e" is very easy to see, but in the future you will save yourself time if you hold the microscope slide to the light and identify the area with the highest concentration of specimens OR, with stained bacteria slides, the area that is stained. Use this information to center your slide on your microscope under the objective lens. The rectangle to the left represents the entire letter e slide. Draw a diagram that shows the orientation of the letter e on your slide when viewed with the naked eye. Front of stage (nearest to you) 11. Start with the lowest powered objective if you cannot find the specimen (or in the case of bacteria, the stained area) with the 4X objective lens, it is unlikely that you ll find it using a higher power since the field of view decreases with increasing magnification. 12. Raise the stage to the top. Note: the 4X objective will not touch the slide. 13. Looking through the oculars, slowly lower the stage using the coarse adjustment until the specimen comes into focus (if you are focusing on the microscope slide, you will see movement when you move the slide). Adjust the light as needed using the light intensity knob and/or the iris diaphragm. 14. Using the stage control knobs, center the specimen in your field of view. 33

34 Microscope Skills 15. Adjust with the fine focus until a sharper image appears (this should require less than one turn of the fine focus knob). Draw a diagram showing the letter e viewed with your 4X objective. Letter e slide diagram 4X objective. Total magnification: Describe the relationship between the orientation of the letter "e" on your prepared slide when you looked at with the naked eye and the image of the e viewed through the compound microscope. 16. With these microscopes, further adjustment of the coarse focus should be unnecessary because these objectives are parfocal (moving the pre-focus knob into the locked position will prevent you from moving the stage up any further, but will not prevent it from being moved down and out of focus). Depth of field decreases with increasing magnification. Depth of field is the distance above or below a specimen that stays in focus. 17. Repeat steps # 13 and # 14 using the 10X and 40X objectives. The 40X will almost come in contact with the microscope slide and cannot be used with some thick mounts. Working distance decreases with increasing magnification. Working distance is the distance between the objective lens and the slide. Letter e slide diagram 40X objective. Total magnification: Describe the relationship between magnification and the amount of the letter e that can be seen through the compound microscope. 18. You may need to adjust the lighting. As magnification increases, the amount of light decreases. 34

35 Microscope Skills Exercise 2b Examining depth of field (crossed threads slide) 19. Change the objective lens back to the one with the lowest power. Lower the stage. Remove the slide with the letter "e" and replace with a slide with crossed threads, which you will use to examine depth of field. 20. Focus on the crossed threads using the 4X objective. Depending on your slide, there will be either two or three threads. Position the image in your field of view so that the point at which the threads actually cross is in the center. Look at the top crossed thread and at the bottom crossed thread. How many threads can you have in focus at once with the 4X objective? Can you focus on both the top thread and the bottom thread at one time? 21. Change to the 10X objective and adjust the focus using the fine focus if necessary. How many threads can you have in focus at once with the 10X objective? Can you focus on both the top thread and the bottom thread at one time? 22. Change to the 40X objective and adjust the focus using the fine focus if necessary. How many threads can you have in focus at once with the 40X objective? Can you focus on both the top thread and the bottom thread at one time? In your own words, describe the relationship between magnification and depth of field. 35

36 Microscope Skills Exercise 3: Field of View and Estimating the Size of a Specimen It is very useful to be able to estimate the size of a specimen you are viewing through your microscope. You can do this easily by comparing the size of your specimen to the total diameter of the field of view. The actual size of the field of view gets smaller as you increase the magnification. So, the first thing you need to know is the diameter of the field of view when you are using each of the different objective lenses. You will use a ruler to estimate the diameter of the field of view for three of the objective lenses on your microscope. You can use the relationship between magnification and relative change in the field of view to calculate the size of the field of view for the 40X and oil immersion lens. A transparent ruler, calibrated in millimeters (cm with 10 mm marks between) can be used to estimate the field of view. Your estimation is less precise than with other measuring devices, such as a stage micrometer, but it should suffice as a starting point. Note: Some microscopes have rulers in the eyepieces. This will NOT help you to estimate the field of view. This kind of ruler will look the same as you change the magnification of the objective it is not actually indicating a size, but is a tool for estimating distances within the field of view. If you have such a ruler on your microscope, ignore it for now. As you progress in your study of biology, you will learn to use this tool. Equipment Needed: 1 compound light microscope per person Clear plastic ruler / ruler piece Procedure: 1. Make sure you understand what is meant by the term field of view. 2. Place the ruler on the stage and focus on it with the lowest power objective lens (4X). Recall that you always start with the lowest power objective. If you are using a small piece of ruler, place it on a blank slide. 3. Line up one of the black mm marks with the left hand edge of your field of view. 4. Count the number of white spaces (mm) visible and record that measurement in the table below. If your microscope has a pointer, you can use that to mark your place, but if not, you so it will be most 5. Repeat steps 3 and 4 using the 10X objective. 36

37 Microscope Skills 6. Repeat steps 3 and 4 using the 40X objective. It may be very difficult to estimate the diameter of the field of view at this magnification. You can estimate it, and use the ruler to see that the estimate is reasonable. Consider that the field of view should be 10 times smaller at a total magnification of 400X than it was at a total magnification of 40X. (Do you see why that makes sense?) 7. What is the diameter of the field of view at a total magnification of 1000X? You will not be able to measure this directly. But think: what was the diameter of the field of view when the total magnification was 100X? Think about the relationship between the increase in magnification and the decrease in the field of view. These numbers will be important as you work on your Microworlds project. 1 mm = µm 1 µm = mm Objective used Total magnification Field of view (mm) Field of view (µm) 4X 10X 40X 100X Notice that you can check to see if you are estimating the diameter of the field of view correctly. As the magnification goes up, the real size of the field of view goes down. Therefore, if you increase the magnification 10 times, the field of view should get 10 times smaller. Check your measurements. 8. Once you have an estimate of the diameter of the field of view, you can put the ruler away. You now have the information about the size of the field of view you need to estimate the size of specimens you are viewing with the same microscope. This will be an important skill for your Microworlds project. 37

38 Microscope Skills Estimating the size of a specimen: Using the 4X objective and a 10X ocular for a total magnification of 40X, the field of view should be about 5000 µm. The specimen (smiley face) fills about half of the field of view and so a reasonable estimate on its size would be 2500 µm. (In other words, how many smiley faces would be required to line up along the diameter? Since it s approximately 2, you would divide the field of view by 2.) 5000 µm? Your instructor may have you practice estimating the size of a specimen found on one of the prepared slides in lab. Choose a slide from your slide set or use the slide specified by your instructor. Specimen name Total magnification Size of field of view µm using the objective lens. Sketch specimen in space provided. Estimate of specimen size µm 38

39 Microscope Skills Exercise 4: Making a Wet Mount Slide of Pond Water Equipment Needed: 1 compound microscope Glass slide Cover slip Pond water Dropper or transfer pipette Procedure: 1. Place a small drop of liquid on a slide. 2. Place a cover slip on slide at a 45 angle, touching to the edge of the drop of liquid. Drop the cover slip gently. 3. For clean-up, carefully remove the cover slip and put in the broken glass container (or the trash if it is a plastic cover slip). Wash and dry the glass slide. Return it to the box of slides. Note: this procedure will be modified if the slide has been used to view blood, any kind of human tissue, or live bacterial cultures. What is the purpose of placing the cover slip on slide at a 45 angle to the drop of liquid and touching to the edge before you drop it? 39

40 Microscope Skills Exercise 5: Wet Mount Slide - Staining Human Cheek Cells using Methylene Blue There are many different types of stains used to make cells or parts of cells more visible. Methylene blue is a very commonly-used biological stain. It is attracted to acidic molecules such as DNA. In this lab exercise, you will remove some of the epithelial cells from your cheek and use methylene blue to see the nucleus more clearly. Equipment Needed: 1 compound microscope per person Gloves Sterile toothpicks Glass slide Cover slip Methylene blue (1 % aqueous solution) 0.9% NaCl Paper towel Safety note: the use of human tissue in this lab requires that you wear gloves and follow your instructor s directions for the safe disposal of lab materials. Procedure: 1. Gather all equipment required. Read through this procedure before beginning. 2. Place a drop of 0.9% NaCl on a glass slide. Set the glass slide down in a safe place on your lab bench. 3. Using a sterile toothpick, gently scrape the inside of your cheek. 4. Place the end of the sterile toothpick that was inside your mouth into the drop of NaCl on the glass slide. Mix gently. Place a cover slip over the mixture using the correct technique for a wet mount. Dispose of the used toothpick in the bleach-filled beaker provided. 5. Place a drop of methylene blue next to the cover slip on your slide. Do not let the stain creep over the top of the cover slip. Be careful; methylene blue will stain clothes and skin. 6. Aid the movement of the methylene blue through the NaCl cheek cell mixture by placing a small piece of paper towel at the opposite edge of the cover slip. Dispose of the paper towel. 7. Place the slide on the microscope and focus on cheek cells beginning with the lowest power objective lens. Increase the magnification until you can see a single cell and the cell nucleus clearly. Most students will find that a total magnification of 400x is the most appropriate. You can draw what you see for your Microworlds project. Using what you have learned about the size of the field of view in Exercise 4, estimate the size of a single human cheek cell: µm = mm Dispose of slide and other lab materials safely, as directed by your instructor. 40

41 Microscope Skills Cleaning and Preparing Scope for Storage: Please keep in mind that the scopes are a shared resource. Your scope may be used by as many as 12 different students in a given week. As a shared resource, it is imperative that each student properly clean and store the scopes to properly maintain the functioning of the scopes. Students must follow each of the following steps before storing the microscope: Cleaning: 1. Rotate the nosepiece to make sure that the lowest power objective (4X) is clicked into position over the stage. (Should be in this position by default when removing the last slide used). 2. Make sure that the stage is gently lowered using the coarse focus knob. DO NOT CRANK DOWN THE COARSE ADJUSTMENT KNOB AS FAR AS IT WILL GO, or you risk damaging the mechanism. 3. Make sure the light intensity knob is at its lowest setting. 4. Turn off the scope. 5. Wet a piece of lens paper with lens cleaning solution. Using gentle pressure, clean each of the objective lenses. 6. When using the oil immersion lens, clean this lens first, and use a fresh lens paper for the remaining lenses. 7. Clean any other lenses, as needed, including the ocular lenses, condenser lens, and light source. 8. Be sure to wipe off any oil or other substances from the stage and other parts of the scope as needed. You also may need to clean any prepared microscope slides that you used. Storage 9. Disconnect the power cord by pulling on the plug, not the cord. 10. Wrap and secure the cord at the back of the scope. 11. Be sure to check that the rotating nosepiece is positioned with the 4X objective pointing toward the base. 12. Cover (if your lab has covers) the microscope and return it to the cabinet. Note carefully how the microscopes should be oriented for the storage cabinet in your lab room. 41

42 Microscope Skills Microscope Study Guide Questions Be able to answer for a quiz. 1. Be able to identify, by name and function, all the parts of the microscope. 2. Define depth of field. 3. Define parfocal. 4. Define resolution/resolving power. What determines the theoretical limits of resolution? 5. Define working distance and field of view. 6. How do you determine total magnification? 7. Which objective lens is used for oil immersion? Can you use that lens without oil? 8. Why does oil immersion technique allow you to see a slide more clearly? 9. How do you properly clean the oil immersion lens? 10. How should the light intensity control knob be set before turning the microscope off/on? 11. What is the procedure for putting a microscope away? 12. What is the correct procedure for focusing on a specimen on a slide? 13. As magnification changes what happens to: depth of field, field of view, working distance, available light? 14. How many microns (micrometers or µm) in a millimeter (mm)? 15. Be able to estimate the size of an object seen in the field of view of your microscope. 42

43 ph and Buffers Lab 3: ph and Buffers Objectives: 1. Define ph. 2. Describe an acidic solution; describe a basic solution. 3. Explain the relationship between hydrogen ion concentration and ph. 4. Explain the use of the ph scale. 5. Explain the use of a ph indicator. 6. Describe the function of a buffer and explain why buffers are important to life. 7. Learn how to use a ph meter. 8. Generate and interpret a titration curve (be able to determine the ph range and buffering capacity). Vocabulary: hydrogen ion, hydroxide ion, acid, base, alkaline, neutral, ph, ph scale, ph indicator, phenol red, buffer, ph range, buffering capacity, anthocyanins, titration, titration curve Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: ph The processes of life take place in an aqueous (watery) environment. Pure water contains equal concentrations of hydrogen ions (H + or protons) and hydroxide ions (OH - ). The addition of certain substances called acids or bases to pure water changes the concentration of these ions. An acid is a substance that donates hydrogen ions (H + ) into a solution. Acidic solutions contain a high concentration of H +. A base is a substance that binds with H +. Some bases, for example sodium hydroxide (NaOH), release hydroxide ions (OH - ) into a solution. The released OH - combines with H +, forming water. Other bases such as ammonia directly bind to H +. In both examples, the base acts to lower the concentration of H + in a solution. Basic solutions are also referred to as alkaline solutions. A neutral solution is characterized by an equal concentration of H + and OH -. Neutral solutions can be made by mixing the appropriate amount of an acid and a base to balance the concentration of each ion. Note that although pure water is neutral, tap water is often not neutral due to the presence of impurities. Due to the process used, distilled water also may not have a neutral ph. ph is a measure of the concentration of hydrogen ions (H + ) in a solution. The ph scale is used to describe the acidity or alkalinity of a solution based on the relative concentrations of H +. The ph scale ranges from 0 ph units to 14 ph units. On the ph scale, 7 is neutral. Values below 7 are acidic, and those above 7 are basic. As the acidity of a solution increases, the H + concentration increases and its ph value decreases. For example, wine, with a ph of 4, is more acidic than milk, which has a ph of 6. The ph scale is logarithmic. This means that for each unit of the scale, 43

44 ph and Buffers there is a tenfold change in the H + concentration. For example, vinegar, with a ph of 3, is 10,000 (10 4 ) times more acidic than pure water, which has a ph of 7. Table 1: ph scale and the ph of some common substances. ph Common Substance 0 strong acid 1 Battery acid 2 Stomach acid, lemon juice 3 Vinegar, beer 4 weak acid Tomato juice 5 Black coffee 6 7 neutral Pure water 8 9 Baking soda 10 weak base 11 House hold ammonia Oven cleaner 14 strong base The ph of a solution can be determined in several ways. A simple way of determining the approximate ph is to use a ph indicator. When placed in a solution, these substances change color depending on the ph. For example, litmus paper contains a ph indicator and when it is dipped into a solution, it will change color. The color of the paper is compared to a set of standards (a range of colors each representing a particular ph). Other ph indicators are added 44

45 ph and Buffers directly to a solution, changing the color depending on the ph. Your instructor may demonstrate the use of the ph indictor phenol red, which turns red in a basic solution and turns yellow in an acidic solution. In this demo, carbon dioxide (CO 2 ) will be bubbled into water containing phenol red. The CO 2 will combine with the water, forming H 2 CO 3 (carbonic acid), which easily releases H +, increasing the concentration of H + in the solution. When enough acid has formed, the phenol red will change color. This reaction between CO 2 and water actually happens in our own bodies: H 2 O + CO 2 H 2 CO 3 H + + HCO - 3 Carbonic acid Bicarbonate ion Our cells release CO 2, which enters our blood stream and is carried to our lungs for exhalation. The CO 2 that enters our blood becomes carbonic acid. Fortunately, our body has a buffering system to prevent our blood from becoming too acidic. You ll be learning about how buffers work as you continue to read today s lab. Another method for measuring the ph of a solution is to use a ph meter. A probe attached to the meter is placed in the solution and the ph is digitally displayed on the meter. The meters used in our lab allow a more precise measurement of ph than a ph indicator. Buffers The biological molecules involved in reactions that take place in the watery interior of the cell and in the solution bathing its exterior are affected by the ph of their environment. Each molecule functions best at a specific ph; even a small change in ph can interfere with their functioning and affect essential physiological processes. For example, if the ph of their surroundings is not optimal, proteins will lose their shape and cease to function. To maintain a constant ph, cells produce buffers. A buffer is a substance that can maintain a particular ph when small amounts of an acid or a base are added to a solution. It does so by donating H + when the H + concentration in a solution is too low or accepting H + if there is too much H + in a solution. Most buffers have two components: a weak acid that releases H +, and its corresponding weak base that binds H +. There are many different buffers and each works within a specific ph range. Some, for example, maintain a ph around 6 while other may maintain a ph near 4. Remember, the role of a buffer is not to neutralize a solution but rather to keep the ph of a solution stable. There is a limit to a buffer s ability to maintain a constant ph. The addition of too much acid or base can overwhelm the buffer s ability to donate or accept enough H + to keep the ph stable. Buffers differ in their buffering capacity. Buffering capacity refers to the ability of a buffer to resist changes in ph with the addition of increasing amounts of acid or base. The more acid or base that can be added before a ph shift occurs, the better the buffer. You will be using a ph meter to find out the buffering capacity of a buffer assigned to your group. 45

46 ph and Buffers 14 ph ml NaOH ml HCl added Figure 1. Titration curve for a solution with no buffering capacity ph ml NaOH added ml HCl added Figure 2. Titration curve for a ph 4 buffer with a moderate buffering capacity. 46

47 ph and Buffers Exercise 1: Red Cabbage ph Indicator Standards Making red cabbage ph standards: In the following exercise you will make a set of ph standards using red cabbage as a ph indicator. The purple color of red cabbage is due to a group of pigments called anthocyanins. These pigments change color depending on ph (figure 3). To make a set of standards, you will set up a rack of tubes containing red cabbage extract, each with a different ph and consequently a different color. Figure 3. Color of cabbage extract at even ph levels. Equipment Needed Per Group: Gloves & safety goggles 5 clean test tubes Grease pencil Red cabbage extract 5 ml pipette for cabbage extract: use the labeled pipette on your tray Pi-pump for 5 ml pipettes Test tube rack Parafilm Buffer solutions, ph 3, 5, 7, 9, 11 47

48 ph and Buffers Procedure: 1. Gather all equipment needed, including gloves and safety goggles, and read through this procedure before beginning. 2. Label five clean test tubes 3, 5, 7, 9, and 11; place tubes in test tube rack. 3. Put on gloves and safety goggles. Into each tube, pipette 5 ml of the buffer that corresponds to the numbered tube. For example, pipette ph 5 buffer into the tube labeled Pipette 3 ml of cabbage extract into each of the tubes. 5. Remove the paper backing from squares of parafilm and stretch a square of the film over the opening of each tube. Holding the film in place with a gloved finger, gently swirl the tube to mix the contents. 6. Describe the color of each tube in Table Save these standards (tubes) for use in Exercise 2. Table 1. ph indicator color data. ph value (tube number) Color of Cabbage Solution

49 ph and Buffers Exercise 2: Determining the ph of Common Household Items You will determine the ph of common substances by adding cabbage extract to tubes of household substances and comparing the color that is produced to the colors in your set of standards. What would be an appropriate control for this experiment? Before you begin, predict the ph of each substance and, following the experiment, review your predictions. Discuss the results with your group and see if you can identify some of the components of the beverages and stomach medications that could have contributed to their ph. Equipment Needed Per Group: Gloves & safety goggles Dropper bottles of the following test solutions: Grape juice Seltzer water 7-up 0.5% sodium bicarbonate (NaHCO 3 ) Maalox Milk of magnesia Vinegar 8 clean test tubes Red cabbage extract 5 ml pipettes: use the labeled pipettes on your tray Pi-pump for 5 ml pipette Set of standards made from cabbage extract (from Exercise 1) Parafilm Procedure: 1. Gather all equipment needed, including gloves and safety goggles, and read through this procedure before beginning. 2. Label each test tube for one of the test solutions. Label one tube as the control. You will need to decide what would be an appropriate control for this experiment. 3. Place 5 ml of each test solution into its labeled test tube. 4. Pipette 3 ml cabbage extract into each tube. 5. Cover each tube opening with parafilm as directed earlier. Gently swirl each test tube to mix the contents. 49

50 ph and Buffers 6. Determine the ph of each test solution by comparing the color of the test solution to the colors of the tubes that make up the set of standards. 7. Record your results in Table 2. Table 2. ph of common beverages and stomach medications. Tube # Beverage or stomach medication tested Predicted ph Determined ph Value 50

51 ph and Buffers Exercise 3: Determining the Buffering Capacity of an Unknown Buffer using the ph Meter Your group will be assigned an unknown ph buffer. Predict the ph range that you think your buffer will maintain. Since you have no real observations upon which to make your prediction, it is just a guess. To this buffer you will do a titration by incrementally adding small amounts of acid, measuring the ph after each addition. You will then do the same with a base, adding small amounts and measuring the ph after each addition. After plotting your data, look at the titration curve (Figure 2) and determine the ph range and buffering capacity of your unknown buffer. Equipment Needed Per Group: Gloves & safety goggles ph meter Unknown buffer 50 ml beaker Bottle of 0.1N HCl (acid) Bottle of 0.1N NaOH (base) Squeeze bottle of distilled water 1 ml pipettes (2): use the labeled pipettes on your tray Pi-pump for 1 ml pipettes 50-mL graduated cylinder (next to each buffer use the cylinder assigned to buffer) Procedure: 1. Gather all equipment needed, including gloves and safety goggles, and read through this procedure before beginning. 2. Use a 50-mL graduated cylinder to measure 20 ml of your unknown buffer into a 50 ml beaker. If there is a letter or code number on the bottle of the buffer you are given, record it in the space provided in case your instructor wants to check your results later. Unknown Buffer Code: 3. Rinse the electrode off with distilled water by holding it over the sink in your lab bench and squirt distilled water over the electrode on all sides. 4. Place the electrode down into the buffer solution and gently swirl. Be careful not to knock the electrode against the beaker. 5. Read the ph value on the digital display and record the value in Table Pipette 1 ml of NaOH into the beaker of buffer. Make sure that you are reading your pipet correctly and ask if you are unsure! You should gently swirl the solution so that the base is well mixed in the solution. Determine the ph as described in steps 4 through 6. 51

52 ph and Buffers 7. Record the ph value in Table 3 that corresponds to 1 ml NaOH added. 8. Continue this procedure, adding 1 ml NaOH, measuring the ph, recording the result, and plotting it on the graph until you have added a total of 10 ml of NaOH. 9. Dispose of your buffer solution as directed by your instructor and wash and dry the beaker. 10. Use a 50-mL graduated cylinder to measure 20 ml of your buffer into the clean 50 ml beaker and repeat the above procedure, but this time use the acid, HCl, instead of NaOH, repeating steps 4-6 above but this time on the side labeled Amount of Acid Added. Be sure to rinse the electrode of the ph meter before you begin. 11. Now repeat the above steps using HCl instead of NaOH. Table 3. ph data for unknown buffer. Amount of Base Added ph of Buffer Amount of Acid Added ph of Buffer Initial ph Initial ph + 1 ml NaOH + 1 ml HCl + 2 ml NaOH + 2 ml HCl + 3 ml NaOH + 3 ml HCl + 4 ml NaOH + 4 ml HCl + 5 ml NaOH + 5 ml HCl + 6 ml NaOH + 6 ml HCl + 7 ml NaOH + 7 ml HCl + 8 ml NaOH + 8 ml HCl + 9 ml NaOH + 9 ml HCl + 10 ml NaOH + 10 ml HCl * Note: ml of HCl indicated is the total amount of acid added * Note: ml of NaOH indicated is the total amount of base added 52

53 ph and Buffers 12. Now graph your data by placing a dot on the graph (Figure 4) at the point where the amount of acid or base added on the x-axis intersects the ph value you recorded. 13. To see the titration curve for your buffer, join the dots on your graph ph ml NaOH added ml HCl added Figure 4. Titration curve of unknown buffer and water 53

54 ph and Buffers 14. Perform a similar titration using distilled water and record your data in Table 4 below. 15. Using your data in Table 4, make marks on your graph (Figure 4) in the same manner as for your assigned buffer. Make sure that you can tell the two graphs apart! 16. Determine the ph range and buffering capacity of your buffer compared to water. Table 4. ph data for distilled water. Amount of Base Added ph of Water Amount of Acid Added ph of Water Initial ph Initial ph + 1 ml NaOH + 1 ml HCl + 2 ml NaOH + 2 ml HCl + 3 ml NaOH + 3 ml HCl + 4 ml NaOH + 4 ml HCl + 5 ml NaOH + 5 ml HCl + 6 ml NaOH + 6 ml HCl + 7 ml NaOH + 7 ml HCl + 8 ml NaOH + 8 ml HCl + 9 ml NaOH + 9 ml HCl + 10 ml NaOH + 10 ml HCl 17. When you are finished dispose of the solution as directed, wash and dry the beaker, rinse the electrode of the ph meter, and put the ph meter in its storage case. Does distilled water act as a good buffer? 54

55 ph and Buffers ph and Buffers Study Guide Questions Be able to answer these for a quiz. 1. What is ph? 2. Explain why ph is such an important factor in the life of an organism. 3. Describe what makes a solution acidic. Describe what makes a solution basic. 4. What is the difference between ph units? (i.e. a solution with a ph of 9 is how many times more basic that a solution with a ph of 8?) 5. Describe the activity of phenol red. 6. Why does water become acidic when you blow into it with a straw? 7. What components of red cabbage extract make it useful as a ph indicator? Why? 8. Describe the function of a buffer. 9. Give an example of a biological buffering system (hint: see challenge question). 10. Explain the difference between a buffer s ph range and its buffering capacity. 11. From a titration curve, be able to determine the ph range and buffering capacity of a buffer. 12. Which has a greater concentration of H+, a solution with a ph of 4 or a solution with a ph of 8? Challenge Question: Explain how the carbonic acid-bicarbonate buffering system contributes to the ph stability of human blood. 55

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57 Biological Macromolecules 57 Lab 4: Biological Macromolecules Objectives: 1. Know the general characteristics of a carbohydrate. Be able to give examples of different kinds of carbohydrates found in both plants and animals. 2. Identify the monomer (s) that make up sucrose and starch. 3. Explain the use of the Benedict s test for reducing sugars and the use of I 2 KI to test for the presence of starch. 4. Describe the structure and function of proteins. 5. Explain the use of biuret reagent for identifying the presence of protein. 6. Explain the use of the paper test for identifying the presence of lipids. 7. Test foods for the presence of reducing sugars, starch, lipids, and protein, and interpret the results of the tests. 8. Explain the use of positive and negative controls in a controlled experiment. Vocabulary: macromolecules, carbohydrates, protein, monomer, condensation, polymer, hydrolysis, monosaccharide, glucose, fructose, galactose, carbonyl group, aldehyde group, aldose, ketone group, ketose, disaccharide, sucrose, polysaccharide, starch, cellulose, chitin, glycogen, lipids, triglycerides, amino acid, peptide bond, Benedict s reagent, reducing sugar, precipitate, I 2 KI (potassium iodide), amylose, amylopectin, biuret reagent, positive control, negative control Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: Four key macromolecules (macro means large in Greek) are the building blocks that form the structure and are involved in the chemical processes of all living things. The four major categories of macromolecules include carbohydrates, proteins, lipids, and nucleic acids. This lab will focus on the first three: carbohydrates, proteins, and lipids. Most macromolecules are formed from smaller molecules called monomers that have been linked together in a condensation reaction to form a much larger molecule called a polymer. Thus, a polymer is a large molecule formed of many monomers joined together. Polymers can be broken apart by hydrolysis reactions. Carbohydrates are macromolecules that serve as building material for cellular structures or as a source of energy. Carbohydrates contain the equivalent of a water molecule for every carbon atom and so are represented as multiples of CH 2 O. For example, glucose with the chemical formula C 6 H 12 O 6 has six carbons and the equivalent of six water molecules. The simplest carbohydrates are the monosaccharides, which are also referred to as simple sugars. Sugars are sweet-tasting small carbohydrates. Monosaccharides, such as glucose, fructose, and galactose, are formed from only one structural unit. Each is characterized by a backbone of three to seven carbon atoms, depending on the sugar. Attached to this backbone are hydrogen atoms (H) and hydroxyl groups (OH) and in some configurations, a carbonyl group

58 Biological Macromolecules (-C=O). The relative placement of these groups gives each sugar its identity and properties. The location of the carbonyl group can be at the end of the molecule, as is the case with glucose, or in the internal structure of the sugar, as seen in fructose (Figure 1). When the carbonyl group is found at the end of the sugar, it is called an aldehyde group and the molecule is called an aldose. When it is found internally, the carbonyl group is referred to as a ketone group and the molecule is called a ketose. In aqueous solutions, simple sugars such as glucose alternate between their linear form and ring form (Figure 2). Glucose: an aldose Fructose: a ketose Figure 1. The linear structure of glucose and fructose. 58 Figure 2. The ring structure of glucose. Simple sugars such as glucose are a major energy source for cells. They can also be joined to formed larger carbohydrates. Disaccharides are carbohydrates formed by the linking of two monosaccharides. Common table sugar is actually a disaccharide called sucrose. It is synthesized by plant cells in a condensation reaction that forms a covalent bond between a molecule of glucose and a molecule of fructose with the loss of a molecule of water. Another

59 Biological Macromolecules disaccharide is lactose, a sugar found in milk that consists of glucose joined to galactose. Polysaccharides are much larger than monosaccharides or disaccharides, consisting of many (hundreds to thousands) monosaccharides linked together to form a long polymer. Polysaccharides can function as energy storage molecules or as molecules for support or structure. Starch is the polysaccharide that serves a reservoir of stored energy in plants. Grains such as wheat and oats are high in starch, as are tubers such as potatoes. Animals store energy in a polysaccharide called glycogen, which is similar in structure to starch but is much more highly branched. Some polysaccharides serve as building material. For example, the cellulose of plants forms their cell wall. The exoskeletons of insects and shellfish contain the polysaccharide chitin. Lipids are the second class of macromolecules explored in this lab. The lipids we will be exploring today are triglycerides. Triglycerides are formed by the linking of fatty acids to a glycerol molecule. Triglycerides, in the form of fats and oils, are often used for energy storage in organisms. Other types of lipids are important for cell membrane structure (phospholipids) or hormones (sterols). Lipids are generally hydrophobic and do not dissolve in water. Proteins are the third class of macromolecules explored in this lab. Proteins are polymers formed by the linking of amino acids by peptide bonds, a type of covalent bond. The twenty naturally occurring amino acids contain a central carbon to which is bonded a hydrogen atom, an amino group, and a carboxyl group (Figure 3). The fourth bond is to a group that is different for each amino acid. This group is commonly referred to as the R group and it is what identifies each amino acid. Each type of protein is characterized by its unique sequence of amino acids. The sequence of amino acids in a protein influences the shape into which a protein naturally folds. The shape of a protein plays an essential role in the functioning of the protein. Proteins have many functions including structural support, speeding chemical reactions, transport, and movement. Amino group Carboxyl group R group: a side chain that differs according to specific amino acid Figure 3. General structure of an amino acid. 59

60 Biological Macromolecules Exercise 1: Preparation of Food Samples Since the foods we eat are or came from formerly living things, we can expect that they will contain macromolecules. Some foods contain all of the macromolecules; some don t. In this exercise, you will prepare some common foods and test them for the presence of these macromolecules. Equipment Needed Per Group: 5 large test tubes Samples of banana, coconut, cream, peanut, and potato De-ionized water Parafilm squares Procedure: 1. Check test tubes for cleanliness. Label the five large test tubes with one of each food type. You will prepare the food samples in these tubes, and use the contents for each of the tests. 2. Preparation of food samples: knives, cutting boards, and mortars and pestles are available for preparing the foods for testing. Solid foods: very finely mince or mash approximately 1 cm 3 of the food and mix in a large test tube with about 10 ml of de-ionized water (avoid using big pieces of food they can get stuck in the pipettes or the test tubes). Liquid foods: put approximately 6 ml into a large test tube. 3. Use Parafilm to seal the tubes before mixing to suspend particles before testing the samples. Note: be sure to use the correct (labeled) knives and other equipment so that you don t contaminate your food samples. Leave the workspace clean for other students. 4. Your food samples are now ready for use in the following tests. 60

61 Biological Macromolecules Exercise 2: Testing for Reducing Sugars Benedict s Test The Benedict's test is used to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). Reducing sugars are capable of reducing (donating electrons to) Cu 2+, which is a component of Benedict s reagent. All monosaccharides are reducing sugars because when heated they convert from a ring form to a linear chain form and their carbonyl group, whether aldehyde or ketone, is free to reduce (donate electrons to) the Cu 2+ with the Benedict s reagent. This causes the reagent to change from transparent blue to a yellow, green, orange, or red precipitate. Note that the color of a positive reaction can vary depending on the substance being tested. Some disaccharides remain in ring form and are only able to reduce Cu 2+ when the bond forming the ring does NOT contain the original ketone or aldehyde groups. If one of these functional groups is available (not bonded), it can then reduce Benedict s reagent and the color change described above is observed. Typically these are sugars with an aldehyde group. Some disaccharides such as sucrose, a ketose, are non-reducing sugars and will not react with Benedict's solution. In this case, the Benedict s reagent remains transparent (Figure 4). Maltose is a reducing sugar Sucrose is a non-reducing sugar Figure 4. Maltose, a reducing sugar and sucrose, a non-reducing sugar. As shown above, many sugars form rings that involve the ketone or aldehyde group. For some sugars, such as maltose, ring formation and ring opening is a reversible process. When the sugar is in its open configuration, the ketone or aldehyde group is available to react with Benedict s reagent. However, for others such as sucrose, the binding of hydroxyl groups locks the ring. These kinds of sugars no longer have a ketone or aldehyde group available to react. 61

62 Biological Macromolecules Equipment Needed Per Group: Gloves & safety goggles Hot plate 250 ml beaker Boiling chips 7 test tubes Test tube rack Wax pencil Dropper bottle of glucose solution Samples of banana, coconut, cream, peanut, and potato Transfer pipet or Pasteur pipet Dropper bottle of de-ionized water Benedict s reagent Test tube holder Procedure: 1. Gather all equipment, including gloves and safety goggles, and read through this procedure before beginning. 2. Make a water bath by filling the 250 ml beaker about a third full of water, adding a few boiling chips. Place the beaker on the hot plate set to highest setting. 3. With a wax pencil, label your test tubes 1-7 (make heavy marks with the wax pencil on the upper part of the tubes so that the heat won t easily melt them off the tubes). 4. Prepare your tubes as indicated in the table below. Tube Food Solution Reagent Total volume 1 2 ml Glucose solution 2 ml Benedict s solution 4 ml 2 2 ml Water 2 ml Benedict s solution 4 ml 3 2 ml Banana 2 ml Benedict s solution 4 ml 4 2ml Coconut 2 ml Benedict s solution 4 ml 5 2 ml Cream 2 ml Benedict s solution 4 ml 6 2 ml Peanut 2 ml Benedict s solution 4 ml 7 2 ml Potato 2 ml Benedict s solution 4 ml 5. Place the tubes in the gently boiling water bath and maintain a gentle boil. 6. After 5 minutes, use a test tube holder to remove the tubes from the water bath. Turn the hot plate to its lowest setting to keep the water bath warm for later use. Place the tubes in a test tube rack. 62

63 Biological Macromolecules 7. Once the tubes have cooled for a few minutes observe the tubes, record your observations and interpret your results. Tube Item Tested Color Reducing Sugars? Which food items contain reducing sugars? 9. Are you surprised by any of your results? 10. What kind of control is Tube 1, a positive control or a negative control? 11. What kind of control is Tube 2, a positive control or a negative control? 12. Why might a sweet-tasting food give a negative result for the Benedict s test? 63

64 Biological Macromolecules Exercise 4: Testing For the Presence of Lipids Paper Test The paper test will be used to test for lipids. This is a very simple test. A small amount of the substance will be applied to a small piece of brown paper and allowed to dry. After this drying time, the presence of lipids can be detected by the translucent appearance of the paper. Equipment Needed Per Group: 7 pieces of brown paper 1 drop of oil 1 drop of water crushed banana, coconut, cream, peanut and potato prepared earlier Procedure: 1. Gather all equipment needed, and read through all of this procedure before beginning. 2. Place a small drop of oil onto one piece of paper - What type of control is this? Place a drop of water onto one piece of paper What type of control is this? For the other items being tested, rub a small amount of the food item directly on the paper, do not use your prepared solutions. 3. Allow samples to dry. 4. Hold each piece of paper up to the light. A translucent look to the paper (light can pass through) indicates the presence of fats or oils. Record your observations and interpret your results. Paper Item Tested Translucent? Lipids Present? 1 Vegetable oil 2 Water 3 Banana 4 Coconut 5 Cream 6 Peanut 7 Potato 64

65 Biological Macromolecules Exercise 5: Testing For the Presence of Starch Lugol s Test (Iodine test) Iodine potassium iodide (I 2 KI) is the reagent used to test for the presence of starch. Starch is formed of two carbohydrates: amylose and amylopectin. Both amylose and amylopectin are polymers made of repeating glucose units. Natural starches contain 10-20% amylose and 80-90% amylopectin. Chains of amylose tend to form coils. When the iodine reagent enters the interior of the coils, it forms a deep blue color. Equipment Needed Per Group: Test tube rack 7 clean small test tubes Dropper bottle of starch solution Distilled water Banana, coconut, cream, peanut and potato solutions prepared earlier I 2 KI iodine reagent (also called Lugol s iodine) Wax pencil Procedure: 1. Gather all equipment needed, including gloves and safety goggles, and read through all of this procedure before beginning. 2. Check test tubes for cleanliness. Label the tubes Prepare your tubes as indicated in the table below. Tube Food solution Reagent Total volume 1 1 ml Starch solution 5 drops I 2 KI 1+ ml 2 1 ml Water 5 drops I 2 KI 1+ ml 3 1 ml Banana 5 drops I 2 KI 1+ ml 4 1ml Coconut 5 drops I 2 KI 1+ ml 5 1 ml Cream 5 drops I 2 KI 1+ ml 6 1 ml Peanut 5 drops I 2 KI 1+ ml 7 1 ml Potato 5 drops I 2 KI 1+ ml 4. Allow reagents to react 1-2 minutes. 5. Observe your tubes and record your observations and conclusions in the table provided. 65

66 Biological Macromolecules Tube Item Tested Color Starch Present? Which food items contain starch? 7. Are you surprised by any of your results? 8. What kind of control is Tube 2, a negative control or a positive control? 9. What type of control is Tube 1, a negative control or a positive control? 66

67 Biological Macromolecules Exercise 5: Identifying Proteins Biuret s Test The use of Biuret reagent is one way to test for proteins. This reagent contains copper sulfate (CuSO 4 ) and potassium hydroxide (KOH). The KOH provides the required alkaline conditions for the reaction. In the presence of peptide bonds, copper ions from CuSO 4 will form a complex with four nitrogen atoms involved in peptide bonds. Copper sulfate solution is normally a bright blue color. When copper sulfate s copper ions are associated with the nitrogen atoms of peptide bonds, the color of the solution changes to a light violet or lavender color. The intensity of the resulting color depends on the size and amount of the protein. The more peptide bonds, the deeper the violet color, while short proteins or small amounts of protein will turn the solution a pink color. Equipment Needed Per Group: Test tube rack 7 clean small test tubes Dropper bottle of egg white (also called egg albumin) Distilled water Banana, coconut, cream, peanut and potato solutions prepared earlier Wax pencil Biuret reagent Parafilm squares Procedure: 1. Gather all equipment needed, including goggles, and read through all of this procedure before beginning. 2. Check test tubes for cleanliness. Label the tubes 1-7 with a wax pencil. 3. Prepare your tubes as indicated in the table below. Tube Food Solution Reagent Total volume 1 2 ml Albumin 1 ml Biuret solution 3 ml 2 2 ml Water 1 ml Biuret solution 3 ml 3 2 ml Banana 1 ml Biuret solution 3 ml 4 2ml Coconut 1 ml Biuret solution 3 ml 5 2 ml Cream 1 ml Biuret solution 3 ml 6 2 ml Peanut 1 ml Biuret solution 3 ml 7 2 ml Potato 1 ml Biuret solution 3 ml 4. Remove the backing from 2 Parafilm squares and stretch one over the lip of each tube. Gently swirl the tubes to mix the contents. 67

68 Biological Macromolecules 5. Let the tubes stand for 2 minutes, then observe the tubes, record the color, and interpret your results. Tube Item Tested Color Protein Present? Which food items contain proteins? 7. Are you surprised by any of your results? 8. What kind of control is Tube 2, a negative control or a positive control? 9. What type of control is Tube 1, a negative control or a positive control? 68

69 Biological Macromolecules Biological Macromolecules Study Guide Questions Be able to answer for a quiz. 1. Describe the structure of a monosaccharide, disaccharide, and a polysaccharide. Give examples of each type of carbohydrate. 2. What are the primary functions of carbohydrates in living organisms? 3. What is the name of a key functional group found in carbohydrates? 4. Explain the use and interpretation of the Benedict s test. 5. Explain why sucrose would test negative using the Benedict s test. 6. If a disaccharide such as lactose tested positive using Benedict s test, what type of sugar must lactose be? 7. Why did we use controls in the tests you conducted for this lab? Explain why water was an appropriate control. 8. Explain the use of I 2 KI (iodine reagent). How would you interpret this test? 9. Describe the structure of a protein molecule. 10. List the functions of proteins in living organisms. 11. Explain the use and interpretation of the biuret test. 12. Be able to name the reagents used to test for the presence of reducing sugars, starch, and protein. 13. What are the limits of these tests? (i.e. what information is not provided by these tests?) Optional Activity (can be done at home): Starch is a storage carbohydrate that is ultimately made up of repeating glucose units. However, it does not taste sweet because the receptors in your taste buds that respond to glucose do not recognize the glucose units when they are polymerized and coiled up, as they are in starch. However, your salivary glands produce an enzyme that hydrolyzes the covalent bonds inside starch, beginning the process of digestion. To demonstrate this for yourself, take a salt-free Saltine cracker and place it on your tongue. Note the taste sensation. Now chew it for 30 seconds without swallowing. Stop and note your perception of the taste. As the enzymes work, the substance should begin to taste sweeter. 69

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71 Diffusion and Osmosis Lab 5: Diffusion and Osmosis Objectives: 1. Describe the role of Brownian motion in diffusion and osmosis. 2. Explain the relationship between simple diffusion and osmosis. 3. Correctly use the oil immersion objective. 4. Explain the differences between simple diffusion, facilitated diffusion, osmosis, and active transport. 5. Predict what will occur when animal cells are placed into solutions that are hypertonic, hypotonic, or isotonic. 6. Predict what will occur when cells with walls are placed into solutions that are hypertonic, hypotonic, or isotonic. 7. Explain the effects of molecular weight on diffusion rate. Vocabulary: semi-permeable, active processes, active transport, bulk transport, passive processes, kinetic energy, Brownian motion, simple diffusion, concentration gradient, solvent, solute, equilibrium, solution, facilitated diffusion, osmosis, osmotically-active substances, osmotic pressure, osmotic concentration, tonicity, isotonic, hypertonic, crenation, hypotonic, lysis, turgid, plasmolysis. Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: The ability of the cell membrane to separate the internal cellular environment from the external environment is essential to cell survival. The cell membrane is the gatekeeper, regulating the flow of substances in and out of the cell. This membrane is semi-permeable; some substances can pass through but others cannot. There are many mechanisms that allow for the passage of substances across the cell membrane. They are categorized based on the source of the energy used. Active processes utilize cellular energy, usually ATP (adenosine triphosphate), and have the ability to move a substance against its concentration gradient. Active transport and bulk transport are processes requiring cellular energy that will be discussed in lecture. Today s lab will focus on passive processes, which do not use cellular energy. Instead, the energy that drives the movement of the substances is the kinetic energy of molecules. The atoms or molecules that make up a liquid or a gas are in constant thermal motion. The temperature of the fluid determines the velocity of these atoms or molecules. As a result of the motion of the molecules, neighboring molecules are hit at random. The impact makes the molecules move. This movement is called Brownian motion and is defined as the random and constant movement of tiny particles when they are suspended in a fluid or gas. This motion is the result of invisible atoms in the fluid bumping the suspended particles and making them jiggle. Today you will observe the effects of Brownian motion on particles of carmine dye suspended in water. 71

72 Diffusion and Osmosis There are several different passive processes powered by Brownian motion that are responsible for the movement of materials across the cell membrane. We will discuss three of these: simple diffusion, facilitated diffusion, and osmosis. Simple diffusion is the net movement of a substance from an area where that substance is in high concentration to an area where that substance is in low concentration. You experience simple diffusion every time you are upstairs and you begin to smell food being cooked downstairs. A concentration gradient is the graduated difference in concentration of a particular substance from one region to another within a fluid or a gas. Because the internal and external environment of the cell is aqueous, substances that leave or enter the cell are dissolved in water. In this case, water acts as a solvent. The substances that are dissolved are called solutes. Important substances like oxygen and carbon dioxide move across the cell membrane by simple diffusion. The chemistry of the cell membrane (a phospholipid bilayer) determines what can pass through by simple diffusion and what cannot. When discussing diffusion, it is important to keep in mind that the movement of substances through a fluid is random. When we talk about something moving down its concentration gradient, we are referring to the net, or overall, movement of a substance from an area of high concentration to an area of low concentration. There will continue to be net movement until there is an equal concentration in both areas, a state called equilibrium. At equilibrium, there is still movement of solute between the two areas, but there is no net movement. Many factors can affect the rate of diffusion, such as steepness of the concentration gradient, electrical or pressure gradients, temperature, and molecular weight of the solute. When the temperature of a solution (solutes dissolved in solvent) increases, it causes increased Brownian motion. This increased motion will allow diffusion to occur more rapidly, and equilibrium will be achieved sooner. The molecular weight of a molecule also affects its rate of diffusion. Small molecules will move at a faster pace than heavy molecules as the same temperature. Think about what would happen if many marbles (solvent molecules) bumped into a bowling ball (a large solute) compared with many marbles bumping into a ping pong ball (a small solute). Facilitated diffusion is the diffusion of a substance across a membrane with the help of a protein. The difference between simple diffusion and facilitated diffusion is the involvement of a protein channel or transporter in facilitated diffusion. Like simple diffusion, the driving force is kinetic energy, and substances only move down their gradients. Substances that are large, charged, or polar do not typically cross the phospholipid bilayer of the cell membrane. These substances are often only able to diffuse through specific protein channels or transporters. Keeping the concentration of glucose in your blood at the right level involves facilitated diffusion. Glucose needs a special protein to help move into a cell from the blood. If your blood sugar gets too high, insulin sends a signal to certain cells to put more glucose transport proteins into the cell membranes. The glucose will move down its concentration gradient, but it can only do so with the right protein to facilitate or help out the process. 72

73 Diffusion and Osmosis Osmosis is the diffusion of water across a semi-permeable membrane. Just like diffusion of other molecules, during osmosis water molecules move from an area of high concentration of water molecules to an area of low concentration of water molecules. Semi-permeable means that some substances will not be able to cross the membrane (osmotically-active substances), but water is able to cross the membrane. It is this inability of some solutes to cross the membrane that allows differences in solute concentrations to occur on either side of the membrane. Osmotic pressure is created by these differences in total concentration of osmotically active substances (osmotic concentration). The osmotic pressure created by the differences in osmotic concentration is a negative pressure that draws the water across the membrane. Since the osmotically-active solutes cannot move to balance out the concentration differences, the water acts to dilute one side to reach equilibrium with the other. So, during the process of osmosis, water moves from an area of low solute concentration to the area of high solute concentration. The ability of a solution to cause a change in the water content via osmosis is called its tonicity. Solutions that have the same osmotic concentration as a cell are considered to be isotonic and will not affect the net water content of the cell. Solutions that have a higher osmotic concentration than a cell are considered hypertonic and will draw the water out of the cell. This loss of water from a red blood cell is called crenation. Solutions that have a lower osmotic concentration than a cell are considered hypotonic, and water will be drawn into the cell. This water movement will cause swelling of the cell, and in cells without a cell wall, can cause lysis or rupturing of the cell. However, cells with a cell wall (plant, fungi, and some bacteria) can resist lysis when put into a hypotonic environment. A plant cell is turgid when in a hypotonic solution. Turgid means that the cell is no longer taking on water because of the mechanical pressure of the cell membrane against the cell wall. A plant can become flaccid or limp if put into an isotonic environment. Plasmolysis will occur if the external environment is hypertonic. The cell membrane pulls away from the cell wall during plasmolysis. You will be performing several experiments that demonstrate the processes of diffusion and osmosis today. You will have several experiments running at the same time. In order to complete all exercises today, you will need to work on several exercises at the same time. It is essential for your group to be organized. 73

74 Diffusion and Osmosis Exercise 1: Permeability of Dialysis Tubing: Water, Glucose and Starch Dialysis tubing is a semi-permeable membrane. The size of the molecule determines its ability to cross this membrane. We will be using different sized molecules (glucose and starch) to observe the properties of this type of membrane and the effects of osmosis. We will be using the Benedict s test to determine the ability of glucose to cross the membrane, the Lugol s iodine (I 2 KI) test to determine the ability of starch to cross the membrane, and any change in the weight of the bag to demonstrate osmosis. Note: Although dialysis tubing is semi-permeable, it does NOT replicate the characteristics of cell membranes. A molecule s ability to cross dialysis tubing is only determined by its size. In a cell membrane size, charge, and polarity of a molecule all affect its ability to cross the membrane. Active transport and facilitated diffusion also affect the ability of a substance to cross a cell membrane. Equipment Needed Per Group: 1 piece of wet dialysis tubing, cm 2 pieces of string or clips 30% glucose solution 1% starch solution 5 ml pipette 5 ml pi-pump 1 ml pipette 1 ml pi-pump De-ionized water ml beakers 3 test tubes Test tube rack Test tube holder Hot plate 500 ml beaker Boiling chips Scale/balance Wax pencil Benedict s solution I 2 KI reagent (Lugol's iodine) 74

75 Diffusion and Osmosis Procedure: 1. Gather all equipment required, and read through this procedure before beginning. 2. Prepare to fill the piece of dialysis tubing with the appropriate solution (see steps 3-5). To do this, first be sure that the dialysis tubing is wet. Then use the string or clip to close one end of the piece of tubing. Fill the dialysis tubing bag with the directed amounts of glucose and starch solution. Rub the top of the tubing between your thumb and forefinger to open the top. Tie off the remaining open end of the tubing, making sure that there are no air bubbles trapped in the bag. The dialysis tubing bag should be limp at the end of this step. 3. Use a pipette to put 5 ml of the 30% glucose solution into the dialysis tubing bag. 4. Use a pipette to add 2 ml of the 1% starch solution to the glucose in the dialysis tubing bag. 5. Rinse the bag with distilled water and use a paper towel to dry the outside of the bag. Do not leave the dialysis tubing bag sitting on the paper towel. Be sure your bag is not leaking. 6. Weigh the bag and record the initial weight in Table 1. You are going to reweigh the bag at the end of the exercise. Why are you weighing the bag? Do you predict a weight change? 7. Add 200 ml of de-ionized water to a 250 ml beaker. Add 20 drops of the Lugol s iodine to the water. Note the initial color in Table 1. Is the test for starch positive? Were you expecting the test to be positive? 8. Put the dialysis tubing bag into the water in the beaker. Make sure the part of the bag containing the solution is completely submerged. 9. Allow dialysis bag to remain in the water for at least 45 minutes. While you are waiting set up exercises 2 and 3. 75

76 Diffusion and Osmosis Exercise 1 continued 10. Prior to removing the bags from the beakers, be sure you have started heating water in the other 250 ml beaker, with boiling chips, on the hotplate. 11. Remove the bag from the beaker. Use a paper towel to dry the outside of the bag. Be sure not to squeeze the bag to dry it, just pat gently with the paper towel. 12. Record the weight in Table 1. Did the weight of the bag change? Did the results match your prediction? 13. Once the bag has been weighed, you can determine the relative permeability of the dialysis tubing to the solutes used in this lab: glucose and starch. You will do this using tests described in Lab 4 (Macromolecules). Use Benedict s solution to test for glucose and Lugol s iodine (I 2 KI) to test for starch. You want to be able to answer two questions: Did the starch move through the dialysis tubing into the water in the beaker? Did the glucose move through the dialysis tubing into the water in the beaker? 14. To check to see if the starch moved, observe the color of the water in the beaker. Note your results in Table 1. The Lugol s iodine is already in the water outside the beaker. Did you get a positive test result for starch? 15. To check to see if the glucose moved from the bag to the beaker, label 3 test tubes: beaker, positive control, and negative control. 16. Put the labeled test tubes in the test tube rack and, using a separate pipette for each, add 2 ml of the solution from the beaker to the test tube labeled beaker. 17. Add 2 ml of the 30% glucose solution to the test tube labeled positive control. 18. Add 2 ml of distilled (or deionized) water to the test tube labeled negative control. 19. Add 2 ml of Benedict s reagent to each of the test tubes and swirl gently to mix. Place each test tube in a boiling water bath for 5 minutes. Carefully remove each test tube from the boiling water with a test tube holder. 20. When cool, observe the test tube for the presence of a yellow, red, orange, or green precipitate. Record your results in Table 1. 76

77 Diffusion and Osmosis Table 1. Results: Permeability of dialysis tubing to water, glucose and starch. Initial Final Weight of Bag Color of Solution in Bag Color of Solution in Beaker Benedict s Test - Beaker Benedict s Test - Glucose (+ control) Benedicts Test - Water ( - control) clear brown brown blue blue Based on these results above, which molecules, if any, were able to cross the dialysis tubing? Complete the table below. Able to Cross Membrane? Direction Moved? Iodine Starch Glucose Water Why did you draw these conclusions? 77

78 Diffusion and Osmosis Exercise 2: Use of Carmine Dye to Demonstrate Brownian Motion Although you will not be able to directly observe Brownian motion, which occurs at a molecular level, you can observe the random motion of microscopic particles in a liquid. Equipment Needed: Compound microscope Slide Cover slip Carmine dye Dissecting needle Dropper bottle of de-ionized water Procedure: 1. Gather all equipment required, and read through this procedure before beginning. 2. Place a few granules of carmine dye in the center of your slide using the dissecting needle. 3. Add a drop of water and cover with the cover slip. 4. View the dye particles using 400x total magnification. 5. Observe the movement of the dye particles. Exercise 3: Observing Plasmolysis in Plant Cells The use of dialysis tubing to observe the behavior of permeable semi-permeable membranes allowed you to observe osmosis in a dialysis tubing bag. This bag is a very simple model cell with no cell wall. The process of osmosis is similar in a cell with a cell wall, but the outcomes can be very different. This exercise will allow you to observe plasmolysis in a plant cell. If time permits draw the plant cell in an isotonic solution for your Microworlds project. You will also be observing plant cells in a hypotonic solution. Equipment Needed: Compound microscope Glass microscope slides Cover slips Aquatic plant leaves Concentrated NaCl solution De-ionized water 78

79 Diffusion and Osmosis Procedure: 1. Gather all equipment required, and read through this procedure before beginning. 2. Prepare a wet mount of a small flat section of an aquatic plant, such as Elodea. 3. Using the compound microscope, search your field for a good example of a cell. 100x total magnification is recommended. Make sure you are able to see a single cell clearly. You may want to draw the cell you observe for your Microworlds project. 4. Without moving your slide, put a drop of concentrated NaCl solution next to the end of the cover slip. Make sure the salt solution does not creep over the cover slip. 5. Aid the movement of the salt solution through the wet mount by placing a small piece of paper towel at the opposite edge of the cover slip. 6. Record your observations in the space provided. 7. Using a new section of leaf, make a new wet mount. Add a drop or two of de-ionized water before putting on the coverslip. 8. Record your observations in the space provided. plant cell pond water plant cell concentrated NaCl plant cell de-ionized water 79

80 Diffusion and Osmosis Exercise 4: Observing the Effects of Hypotonic and Hypertonic Solutions on Red Blood Cells You will be using isotonic, hypertonic, and hypotonic solutions to observe the effects these solutions have on the shape of red blood cells. Blood cells are very small cells, and you will need to use your oil immersion lens to observe the effects of these solutions. Be sure to observe the red blood cells in isotonic solution first so that you know what these cells look like normally. Be aware that in the hypotonic solution, it is possible that you will not see cells or that may seem to disappear while you are observing them. This is due to lysis. If time permits, draw the blood cell in an isotonic solution for your Microworlds project. Equipment Needed: Gloves Dropper bottle of sheep blood 3 slides 3 cover slips Compound microscope Dropper bottle of 0.9% NaCl (isotonic solution) Dropper bottle of concentrated NaCl (hypertonic solution) Dropper bottle of distilled water (hypotonic solution) Wax pencil Immersion oil Lens paper Lens cleaner Safety Note: the use of blood in this lab requires that you wear gloves and follow your instructor s directions for safe disposal of lab materials. Use of Oil Immersion Objective Oil immersion objectives are designed to work best with oil as a medium for the light path. Light bends less when it passes through oil compared to air, so more light traveling from the specimen on the slide actually gets to the objective lens. This provides better resolution for smaller objects. Any objective lens with a magnification of 90X or greater is an oil immersion lens. Therefore, you won t see very much if you try using the 100X objective lens on your microscope with nothing but air between the lens and the slide. The field of view is so small that not enough light is coming from the specimen to the lens to form a good image. 80

81 Diffusion and Osmosis Oil immersion objective oil oil Procedure: 1. Focus on your slide starting with the scanning objective and work your way through the lenses up to high power (40X). It is critical that your specimen is in focus before proceeding to the oil immersion lens. 2. Without moving the fine focus knob, rotate your nosepiece so that the 40X objective and oil immersion lens are on either side of your specimen (see illustration above). 3. Place a small drop of immersion oil on the slide at the center of the specimen. 4. Move the 100X objective into place it will come in contact with the oil and cover slip. 5. Taking care not to move the coarse focus up or down, focus with the fine focus knob only. 6. ALWAYS clean up after using the oil immersion objective: a. Turn light intensity knob down and switch the microscope off. b. Move objectives so the specimen is again between the 40X and 100X. c. Lower the stage and remove the slide. d. Move the 100X objective toward the front and wipe all of the oil off the 100X lens. Use ONLY lens paper on the oil immersion lens. Note: use of ANY other materials may permanently scratch the lens! e. Using another section of lens paper and a few drops of lens cleaner, thoroughly clean the oil immersion objective lens. Use another clean section of lens paper to dry the lens. Repeat this process until all oil is removed from the objective. f. Clean the oil off of the slide. You should use lens paper for slides. g. Using a lens paper, clean the remaining objective lenses and the ocular lenses. Be sure to check the stage and other components for oil and clean appropriately. 81

82 Diffusion and Osmosis Effects of Hypotonic and Hypertonic Solutions on Red Blood Cells Procedure: 1. Gather all equipment required, and read through this procedure before beginning. 2. Place a small drop of blood on a slide. A touch of the dropper to the slide will be enough do not put an entire drop on the slide 3. To the slide add a drop 0.9% NaCl to the drop of blood. Place a coverslip on your sample. 4. Use the compound microscope to view the blood cells in isotonic solution. Due to their small size, you will need to use oil immersion to view these cells. Be sure to review the procedure as given previously. Record your observations in the space provided 5. After viewing under oil immersion, rotate the nosepiece so the oil immersion lens and scanning (4x) objective are on either side of the slide. Carefully add a small drop of 3% NaCl to one edge of the coverslip, and use a filter paper (kimwipe or paper towel) to draw the solution over the red blood cells. After adding the new solution, rotate the oil immersion lens back into position and observe your cells. Record your observations in the space provided 6. To view the cells in the hypotonic solution, follow the directions as above, but instead use de-ionized (distilled) water. Be sure to focus on the cells in a timely manner. Record your observations in the space provided red blood cell 0.9% NaCl red blood cell concentrated NaCl red blood cell de-ionized water 82

83 Diffusion and Osmosis Optional Demonstration: Observing the Effects of Molecular Weight on Diffusion Rate You will be comparing the diffusion rates of two molecules with different molecular weights. Equipment Used: Agar plate Ruler Straw Tweezers Dropper bottle of 1.5% potassium permanganate (KMnO 4 ) Dropper bottle of 3.5% methylene blue (C 16 H 18 ClN 3 S) Procedure: 1. Before observing the experiment prepared for you, answer the following questions: a. Using a periodic table, determine the molecular weight of potassium permanganate (KMnO 4 ) and record it below: b. Using a periodic table, determine the molecular weight of methylene blue (C 16 H 18 ClN 3 S) and record it below: c. Predict which molecule will diffuse faster. 2. Two wells have been created in the agar plate using a straw to remove two equally sized plugs of agar. 3. One small drop of the potassium permanganate solution was added into one well. 4. One small drop of methylene blue was added into the other well. 5. Record the distance each stain has diffused into the agar. Potassium permanganate mm Methylene blue mm 6. Compare the results to your predictions. 83

84 Diffusion and Osmosis Diffusion and Osmosis Study Guide Questions Be able to answer for a quiz. 1. What is the driving force (energy) that powers passive processes? 2. What is the driving force (energy) that powers active processes? 3. What does it mean for a membrane to be semi-permeable? 4. What is diffusion? 5. How are simple diffusion and facilitated diffusion different? 6. What is osmosis? 7. How would you describe a solution that is hypotonic, hypertonic, or isotonic to the contents of the cell? 8. How do cells with cell walls differ from cells without cell walls, when put in a hypotonic environment? 9. Understand the terms: turgid, flaccid, plasmolysis, lyse, and crenate, with respect to osmosis in cells. 10. Is osmosis an example of an active or passive process? 84

85 Enzymes Objectives: Lab 6: Enzymes 1. Understand what an enzyme is and how it functions in a reaction. 2. Identify the substrates and products of the catecholase-catalyzed reaction. 3. Become familiar with the lab procedure and measure the rate of benzoquinone production. 4. Use your understanding of enzymes and protein structure to predict the effect of ph, temperature, salt concentration, enzyme concentration, and substrate concentration on enzyme activity. 5. Use the knowledge gained from observations of the initial procedure to develop a hypothesis involving another independent variable of your choice. Design and carry out an experiment to test the new hypothesis. 6. Learn how to graph and interpret your data. Vocabulary: enzymes, catalysts, activation energy, reactants, substrate, active site, transition state, product, salt concentration, ph, enzyme concentration, substrate concentration, temperature, catecholase, catechol, benzoquinone, spectrophotometer, absorbance, transmittance, cuvette, rate of reaction Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: Enzymes are protein catalysts present in all living organisms. A catalyst lowers the activation energy and allows reactions to proceed at a faster rate. The activation energy can be thought of as the initial push that the reaction needs to get going. The exact mechanism by which an enzyme accomplishes this can be very different depending on the type of enzyme. A chemical reaction has reactants (what you start with) and products. Substrates are the specific reactant molecules that bind to a specific part of the protein called the active site. The reactants and enzymes are both 3-dimensional and must fit together precisely, which allows a great deal of specificity. For example, the enzymes that can break the bonds in lactose cannot break the bonds in maltose, even though both are dissacharide sugars. When substrates bind to the active site of an enzyme, a shape change may occur for both participants. This shape change may help the substrates reach the transition state. In a chemical reaction, the transition state is an unstable and high-energy configuration that the substrates have to achieve before they can be altered to become the products. The energy required to get the substrates to the transition state is the activation energy. Enzymes lower this activation energy and help the chemical reaction occur at a faster rate. Once the reaction is complete, the product is formed and the enzyme returns to its original state, ready for another round of catalysis. Enzymes (as with all catalysts) are not used up during the course of a reaction. 85

86 Enzymes Many factors may affect the rate of enzyme activity. Factors that can affect protein folding (remember that enzymes are proteins and that their shape is critical to their function) include salt concentration (this really means ion concentration; think about what happens to a salt in an aqueous solution), ph, and temperature, as well as other small molecules. Each enzyme has an optimal range of ph, temperature, and salt concentration for its functions. Most human enzymes have the highest activity between ph 6-8. This is one of the reasons why it is critical for living organisms to maintain an internal environment within a particular range of parameters (homeostasis). Without this control, the environment would not be optimal for enzyme function, and metabolic processes will be negatively affected. The enzyme concentration and substrate concentration can also influence the rate of metabolic processes. Adding more enzymes may increase the rate of the reaction, but the rate will decrease once all substrate molecules are used up. The addition of more substrate can also increase the reaction rate initially but the rate will level off once all the enzymes have been saturated with substrate. Temperature can influence enzyme-catalyzed reactions in two different ways: 1) by affecting the shape of the enzyme, and 2) by increasing the energy level of the substrates (Pitkin 1992). The optimal temperature for most enzymes found in the human body is 37 C. Think about how this relates to what happens to a person when they get hypothermia (low core body temperature). In this exercise you will use the enzyme catecholase from a potato. The substrates are a colorless compound called catechol and molecular oxygen. The potato cells synthesize the enzyme catecholase as well as one of its substrates, catechol. In undamaged cells, catecholase is stored in vesicles within the cytoplasm therefore; it does not interact with catechol. When the potato cells rupture, as happens when a potato is cut, the cytoplasm (or interior of the cell) is exposed to the oxygen in the air. Catecholase now has both of its substrates available in the presence of the enzyme and the reaction can proceed. Benzoquinone is the reddish-brown product of the reaction. Although the potato cells make their own catechol, we will be adding more to make sure the substrates are not limiting. Catecholase (enzyme) Catechol + O 2 (colorless) Benzoquinone (reddish-brown) Catecholase is a term that represents a group of enzymes (like tree refers to a group of different plants) and has some synonyms: catechol oxidase, tyrosinase (a closely related enzyme; the term was once used as a synonym), and polyphenol oxidase (a broad term that includes both catechol oxidases and tyrosinases). A color chart (Figure 1) can be used to indirectly determine the rate of the reaction. Samples are compared with the color chart to determine the number for color intensity that matches the sample most closely. The darker and more intense the color, the more benzoquinone that has formed. 86

87 Enzymes Figure 1. Color chart for the catecholase-catalyzed reaction. Exercise 1 - Estimating Rate of Product Formation Using a Color Chart You will observe and measure the production of benzoquinone over a given amount of time. You can use this information to calculate the rate of the reaction. You will determine the rate of your reaction by graphing your color chart data with respect to time, and calculating the slope of the line (Figure 2). Don't be discouraged if your data do not look as straight forward as Figure 2. The first exercise will also allow you to become familiar with the equipment and the procedure for following this reaction. Think about what variables might affect the rate of this reaction. 87

88 Color Intensity BI 112 Enzymes Color Intensity v. Time run 1 run 2 run Time (minutes) Figure 2. Using slope to determine reaction rate. In this experiment, you must calculate slope to estimate the rate of reaction for each of your trials. To calculate slope, simple divide the rise of the line/the run of the line. For example, when looking at Figure 2, you can see that each run in the experiment produces lines with different slopes. For run 3, the slope is most easily calculate by using the portion of the line from 0 to 2 minutes (rise = 4, run = 2, slope = rise/run = 4/2 = 2), after that, the reaction has gone to completion and is at equilibrium (the intensity will not increase). For run 2, the reaction goes to completion at 4 minutes, so the slope of run 2 is 1 (rise/run = 4/4). For run 1, since it does not go to completion, we can estimate the slope by making a best fit line and calculating the slope, which is 0.4 (rise/run = 2/5). So, regardless of the shape of your line in your graph, you will be able to calculate slope. Keep in mind; you will be studying the RATE of the catecholase reactions in this experiment, so slope MUST be calculated for each trial. 88

89 Enzymes Exercise 1: Catecholase and Rate of Reaction Equipment Needed Per Group: Gloves & safety goggles 3 test tubes Grease pencil Stopwatch 1 ml pipette for potato extract: use the labeled pipette on your tray 5 ml pipette for catechol: use the labeled pipette on your tray 5 ml pipette for distilled water: use the labeled pipette on your tray Pi-pump for 5 ml pipette Pi-pump for 1 ml pipette Test tube rack Bottle of distilled water Potato extract 0.05% catechol Color chart Parafilm and scissors Procedure: 1. Gather all equipment required, including gloves and safety goggles. Read through this procedure before beginning. 2. Look at Table 1 to see how the experiment has been designed, and answer the following questions. Table 1. Reaction mixtures for the catecholase experiment. Tube 1 Tube 2 Tube 3 Water (ml) Catechol (ml) Extract (ml) a. What hypothesis is being tested? b. What is the independent variable in this experiment? 89

90 Enzymes c. What is the dependent variable? d. Is there a control for this experiment? If so, what type of control is it? e. Should a reaction be occurring in Tube 1? Tube 2? Tube 3? f. Which tube would you predict would have the fastest reaction rate? WHY? 3. Get three large test tubes and label them 1, 2 and 3 with a grease pencil (these tubes will contain the solutions outlined in Table 1). 4. Use the provided pipette to measure the water into tubes 1, 2, and 3 (see Table 1). 5. Use the appropriate pipette to add 2 ml of catechol to each tube. 6. Use the provided pipette to add potato extract into tubes 1, 2, and 3 (see Table 1). 7. Place parafilm tightly over the top of each tube and invert the tubes to mix their contents. 8. Immediately after adding the potato extract, observe the intensity of the colors (the intensity is more important than the actual color). In Table 2, record the number of the color intensity that most closely matches the color intensity of each tube for Time = 0 min. 9. Shake each tube gently at 1-minute intervals to keep the reactants well-mixed. 10. Observe the color intensity of your sample every minute for five minutes. Record your observations in Table 2. 90

91 Color Intensity BI 112 Enzymes Table 2. Results of catecholase experiment: color intensity over 5 minutes. Tube 1 (1 ml extract) Tube 2 (0.5 ml extract) Tube 3 (0 ml extract) 0 min 1 min 2 min 3 min 4 min 5 min 11. Using the data from Table 3, draw three graphs illustrating the change in color/intensity over time. For each graph, estimate the slope of the line. The slope of each line in your graph represents the reaction rate for each tube in the experiment. 5 Figure 3. Graph of results from Table 3. Color Chart Intensity v. Time Time (minutes) 12. Calculate and compare the slopes for each tube: Tube 1 slope Tube 2 slope Tube 3 slope Was your hypothesis supported by the results? 91

92 Enzymes Questions 1. Predict the color change of a tube containing 3.75 ml of water, 2 ml of catechol, and 0.25 ml of potato extract. Explain how you derived this prediction from your data. 2. Why is it necessary to add the potato extract to each tube last, after the water and catechol are already measured? 3. Why was a different amount of water added to each tube? 92

93 Enzymes Exercise 2: Designing Your Own Experiment Your initial observations may lead to you to ask some questions about how altering the conditions of the experiment might alter the rate. Choose one independent variable (one condition to alter; see below) and using your knowledge of enzymes and proteins in general, generate a hypothesis. Design an experiment to test that hypothesis as a group. See your instructor for a list of additional equipment that is available. Available Materials May Include: ph solutions: ph 3, 5, 7, 9, 11 Saline solutions: 0.9% NaCl, 5% NaCl, 10% NaCl, 25% NaCl Carbohydrates: 1M fructose, 1M sucrose, 1% starch Water baths: ice (0 C), room temperature (~22 C), 37 C, 60 C, boiling (100 C) Literature Cited: Pitkin, R Enzyme investigations for introductory courses. In: Goldman, C.A. (ed). Tested studies for laboratory teaching, Vol. 13. Proceedings of the 13th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), pp.191. Record your experimental design and setup in table 3 below. What is your Independent Variable? What controls/constants will you use? Table 3. Experimental Design for Self-Guided Study Reagents Used Tube 1 Tube 2 Tube 3 93

94 Enzymes Record your observations in Table 5. Table 4. Results of self-guided catecholase experiment: color intensity over 5 minutes. Tube 1 Tube 2 Tube 3 Replicates Replicates Replicates A B C A B C A B C 0 min 1 min 2 min 3 min 4 min 5 min Table 5. Averages of self-guided catecholase experiment: color intensity over 5 minutes. Tube 1 Tube 2 Tube 3 0 min 1 min 2 min 3 min 4 min 5 min 94

95 Color Intensity BI 112 Enzymes Using the data from Table 5, draw graphs illustrating the change in color/intensity over time. Figure 4. Graph of results from Table 4. 5 Color Chart Intensity v. Time Time (minutes) From the lines above, calculate the slope for each of your tubes. Tube 1 slope Tube 2 slope Tube 3 slope Was your hypothesis supported by the results? 95

96 Enzymes Enzymes Study Guide Questions Be able to answer for a quiz. 1. For the chemical reaction studied this week, what are the substrate(s)? The product(s)? 2. Be able to recognize controls in your experiments. 3. Be able to interpret graphs of data. 4. In general, are most enzymes specific? What makes this possible? 5. Enzymes belong to which group of macromolecules (biological molecules)? 6. Name at least 3 factors (variables) that may affect enzyme activity. 7. What is the optimal temperature range for most human enzymes? Would you expect all enzymes to have the same optimal temperature range? 8. What is the optimal ph range for most human enzymes? Would you expect all enzymes to have the same optimal ph range? 9. What is the effect of increasing the amount of substrate? 10. What is the effect of increasing the amount of enzyme? 11. What is the relationship of a catalyst to the activation energy? 12. The reaction that you have observed in lab today is a common one that occurs in many cells. Can you use what you have learned to explain why raw potatoes turn brown if left on the table but cooked potatoes do not? 13. Can you use what you have learned today to explain why a common ingredient for fruit salad is often lemon juice or some other kind of citrus juice? Challenge Question: Use the internet or a similar resource, do some research about the process of tanning in humans. Use the key words: tyrosinase, tyrosine, melanin, and melanocytes. Could you explain how someone s skin changes color when they tan using what you have learned about enzymes in lab? Challenge Question: Could you explain what happens when your blood clots after you cut yourself? Focus on what missing piece of the chemical equation has been added. Use the vocabulary introduced in this lab: substrate, enzyme, and product. 96

97 Fermentation Lab 7: Fermentation and Cellular Respiration Objectives: 1. Relate the activity of enzymes to the metabolic pathways used in catabolism. 2. Differentiate between aerobic respiration, anaerobic respiration, and fermentation. 3. Explain how the production of CO 2 by fermenting yeast cells can be measured. 4. List the environmental factors that can affect enzyme activity and relate this to the rate of fermentation. 5. Use the knowledge gained from observations of the initial procedure to develop a hypothesis involving another independent variable of your choice. Design and carry out an experiment to test the new hypothesis. Vocabulary: metabolism, energy, catabolic reactions, ATP (adenosine triphosphate), substrate, metabolic pathway, aerobic respiration, anaerobic respiration, fermentation, final electron acceptor, aerobes, glycolysis, Kreb s cycle, electron transfer phosphorylation, anaerobes Microworlds Opportunity: Check with your instructor regarding any microworlds slides you may use in this lab. Introduction: Metabolism is a term that refers to all the chemical reactions taking place in a cell. In this lab, you will be focusing on those reactions involved in the release of energy. All organisms must capture the energy stored in large molecules to fuel life processes. Large molecules are broken down into simpler ones, releasing the energy stored in the chemical bonds that held the larger molecules together. Plants and certain protists and bacteria are able to capture energy from the sun, and use that energy to reorganize atoms of C, H and O into glucose, C 6 H 12 O 6. Glucose may be used as a starting point in the synthesis of other organic molecules. When organisms need energy to carry out other reactions, they break down glucose (or molecules that are made from glucose) in order to release that energy. The breakdown of glucose is central to the metabolism of all organisms. Even plants, which make glucose, use it as energy storage, and break it down to release usable energy. Reactions that release energy by breaking apart complex molecules into smaller molecules are called catabolic reactions and are exergonic. Very often the energy captured in catabolic reactions is not directly used but instead is transferred to an energy storage molecule called ATP (adenosine triphosphate). The energy stored in ATP is released as needed to fuel many processes including the transport of certain substances across cell membranes, movement, and the synthesis of cellular structures. Additionally, many chemical reactions require an input of energy to get the reactions started (i.e. they are endergonic). 97

98 Fermentation Catabolic reactions often require the assistance of enzymes to lower the amount of activation energy needed to get the reactions to occur rapidly enough to meet the needs of the cell. Energy releasing processes occur as a series of steps, each step catalyzed by a different enzyme. The product of one step becomes the substrate for the enzyme of the following step. Recall that a substrate is the molecule that a specific enzyme acts upon. A series of enzymecatalyzed reactions involved in a cellular process is called a metabolic pathway. Environmental factors such as ph, salt concentration, and temperature can affect enzyme activity and thus can change the rate of a metabolic pathway. The concentration of enzymes and substrate also influences the rate of metabolic processes. Adding more enzymes may increase the rate of the reaction but the rate will decrease once all substrate molecules are bound by all available enzymes. The addition of more substrate can also increase the reaction rate initially but the rate will level off once all the enzymes have been saturated with substrate. Living things are amazingly diverse in the types of molecules they can degrade for energy. They also vary in the ways they obtain energy. There are three main processes by which organisms can obtain energy: aerobic respiration, anaerobic respiration, and fermentation. Aerobic respiration involves a series of enzyme-catalyzed reactions resulting in the breakdown of glucose into CO 2 (carbon dioxide) and H 2 O, the production of approximately 36 to 38 ATP molecules, and the use of O 2 (oxygen) as the final electron acceptor. Organisms that use oxygen as the final electron acceptor for respiration are called aerobes. You may recall from lecture that aerobic respiration occurs via glycolysis, the citric acid cycle, and electron transfer phosphorylation. Carbohydrates other than glucose can be modified and converted to glucose, which can then enter glycolysis. Other energy sources such as lipids and proteins are also modified and enter the pathways of aerobic respiration at various points. Plants, animals (including people!), fungi, most protists, and many bacteria use aerobic respiration to capture energy. Below is the summary equation of aerobic respiration. It shows the starting reactants and the products, but not the many reactions in between. C 6 H 12 O 6 (glucose) + 36ADP + 36P i + 6O 2 6CO 2 + 2H 2 O + 36ATP Certain bacteria, called anaerobes, can obtain energy by anaerobic respiration. These bacteria respire via glycolysis, the citric acid cycle, and electron transfer; however they use other molecules such as nitrate (NO 3 - ), nitrite (NO 2 - ), or sulfate (SO 4 2- ) rather than oxygen as their final electron acceptor. These are only a few of the many possible electron acceptors that can be used. This ability allows anaerobic bacteria to respire in places where oxygen is unavailable. Unlike aerobic respiration, which breaks glucose and other carbon compounds down to CO 2 and H 2 O, fermentation is a metabolic process that partially degrades energy sources. Fermentation does not use O 2 as a final electron acceptor; instead the pyruvate (or a modification of pyruvate) produced at the end of glycolysis receives the electrons from glucose (and is thereby reduced), becoming a waste product. A small amount of ATP is produced, 98

99 Fermentation typically 2 molecules of ATP for every molecule of glucose. Even though this is not very much energy, cells are able to re-oxidize NADH to NAD +, an essential coenzyme for glycolysis. Without fermentation, there would be no NAD + available and even glycolysis would not occur. The kind of waste product formed in fermentation depends on the organism. The kinds of fermentation enzymes an organism can make determine the type of waste produced. There are many different possible waste products, including alcohols such as ethanol or propanol, and acids such as lactic acid and acetic acid. Organisms that are capable of fermentation include yeasts (a type of single celled fungus), bacteria and your muscle cells. Fermentation also occurs in many organisms that typically use aerobic respiration, called facultative anaerobes. In these organisms, when oxygen levels are too low to allow the electron transfer chain to function, fermentation occurs. Yeast, the subject of today s lab, are used in the production of many food products, such as bread, wine and beer. The fermentation pathway used by yeast cells produces ethanol (a type of alcohol) and CO 2 as waste. When making bread, yeast cells in the bread dough ferment the carbohydrates in the dough. The CO 2 (which is a gas) they produce makes the dough rise. The ethanol that is made is driven off when the bread is baked. Below is the summary equation for the fermentation process used by yeast cells: C 6 H 12 O 6 (glucose) + 2ADP + 2P i +2NADH 2CO 2 + 2C 2 H 5 OH (ethanol) + 2ATP +2 NAD + In muscle cells, and in some bacteria, the pyruvate produced at the end of glycolysis is reduced directly to lactic acid. How do you think this relates to the burning feeling you get in your muscles when you are breathing hard? How do you think this relates to yogurt? The summary equation for lactic acid fermentation is below. C 6 H 12 O 6 + 2ADP + 2P i +2NADH 2CH 3 CHOHCOOH (lactic acid) + 2ATP +2 NAD + 99

100 Fermentation Exercise 1: Alcoholic Fermentation by Yeast Cells Guided Study In this exercise you will mix yeast cells with a glucose solution. As the yeast ferments the glucose, ethanol and CO 2 are formed. The rate of fermentation will be determined by measuring the amount of CO 2 produced over time. Equipment Needed Per Group: Gloves & safety goggles 3 fermentation tubes (careful-very expensive) Yeast suspension 30% glucose Parafilm squares 5 ml pipettes Pi-pumps Wax pencils Procedure: 1. Gather all equipment needed including gloves and safety goggles, and read through this procedure before beginning. 2. Make a prediction of your results, and from your prediction create a hypothesis. Identify the independent variable and dependent variable of the experiment. Now test your hypothesis by running the experiment. Hypothesis: Independent variable: Dependent variable: 100

101 Fermentation 3. Collect 3 fermentation tubes. Label them number 1, 2, and 3. Figure 1. Fermentation tubes. 4. Using the 5 ml pipettes provided, being careful not to contaminate solutions, set up your tubes using the volumes listed in Table 1. One of the tubes will be the negative control (make sure you know which one!). The other two tubes differ in the volume of yeast suspension. The more yeast solution in the tube, the greater the amount of enzymes. That is because it is the yeast that synthesizes the enzymes! Table 1. Contents of fermentation tubes. Fermentation Tube Reagents Water (ml) Yeast suspension (ml) % glucose (ml) Place a square of parafilm over the opening of each tube. Mix the fermentation tube solution, invert it (long tube away from you), and allow the solution to fill the long tube. Turn the tube back to its original position (Figure 1). The long tube should be completely filled with fluid. Make a baseline mark at the top of the tube where the sides are straight rather than curved. Remove the parafilm from all tubes. 101

102 Fermentation 6. Check with your instructor to see if you should incubate your tubes in a 37 C water bath. 7. At 5 minute intervals, measure in mm and record the distance from the baseline mark to the fluid level in Table 2. If a space forms with many bubbles, take the reading at the interface of the liquid and the bubbles, not the top of the bubbles. Continue taking data for 20 minutes. 8. Special clean-up instructions: rinse fermentation tubes thoroughly; leave a little clean water in each tube to soak. Wipe tables thoroughly. Table 2. CO 2 displacement in fermentation tubes (mm). Fermentation Tube minutes 10 minutes 15 minutes 20 minutes 9. Complete the graph/grid below to determine the rate of fermentation for each of your tubes. 102

103 CO 2 (mm) BI 112 Fermentation CO 2 Displacement v. Time Time (minutes) Tube 1 slope Tube 2 slope Tube 3 slope 10. Use your data to determine whether or not your hypothesis was supported. Which tube was the control? What was the independent variable? What is the dependent variable? What was your hypothesis? Did the data support your hypothesis? 103

104 Fermentation Exercise 2: Fermentation - Design Your Own Fermentation Experiment Design an experiment based on the yeast fermentation exercise. For your own experiment, you will choose one variable to alter. Note: remember to keep the total volume in your fermentation tubes at 14 ml. Available materials: ph solutions (substitute for H 2 O to alter the ph): ph 3, 5, 7, 9, 11 Saline solutions: 0.9% NaCl, 5% NaCl, 10% NaCl Carbohydrates: Monosaccharides: 1M fructose, 1M glucose, 1M galactose Disaccharides: 1M sucrose, 1M lactose Polysaccharides: 1% starch Sugar substitutes (see Figure 2):1M saccharin, 1M aspartame Water baths: ice (0ºC), room temperature (~22ºC), 37ºC, 60ºC, boiling (100ºC) Splenda (sucralose) Sweet-n-Low (saccharin) Figure 2. Chemical structure of some common sugar substitutes. (Hint: Are the structures similar to glucose (see Figure 3) or very different?) Figure 3. Chemical structure of glucose. 104

105 Fermentation Create a hypothesis and identify the independent variable and the dependent variable. Then design and run an experiment to test your hypothesis. Record your observations in the provided chart(s) Hypothesis: Independent variable: Dependent variable: Experimental design: Table 3. Reagents used in group-designed experiment Fermentation Tube Reagents (ml) Water Yeast Carbohydrate 105

106 Fermentation Table 4. Results for group-designed experiment Fermentation Tube Number Replicates A B C A B C A B C 5 minutes 10 minutes 15 minutes 20 minutes Table 5. Averages for group-designed experiment Averages for Replicates Fermentation Tube Number minutes 10 minutes 15 minutes 20 minutes 106

107 CO 2 (mm) BI 112 Fermentation Complete the graph/grid below to determine the rate of fermentation for each of your tubes. 30 CO 2 Displacement v. Time Time (minutes) Tube 1 slope Tube 2 slope Tube 3 slope Use your data to determine whether or not your hypothesis was supported. Which tube was the control? What was the independent variable? What is the dependent variable? What was your hypothesis? Did the data support your hypothesis? 107

108 Fermentation Fermentation and Aerobic Respiration Study Guide Questions Be able to answer for a quiz. 1. Why do all cells need energy? 2. What molecule (that acts as an energy storage molecule) is formed as a result of catabolic processes? 3. What does the term catabolism mean? 4. Explain how the processes of aerobic respiration, anaerobic respiration, and fermentation differ. In what ways are they similar? 5. Identify the final electron acceptor used for aerobic respiration, anaerobic respiration, and fermentation. 6. Is glucose the only source of energy? Explain your answer. 7. What is meant by a metabolic pathway? 8. Describe the role of enzymes in catabolism. 9. Identify the environmental factors that can affect enzyme functioning. 10. What are the waste products formed during fermentation by yeast cells? 11. Explain how the rate of alcoholic fermentation can be measured. Challenge Question: Some sources suggest that corn-fed or grain-fed cattle require more antibiotics to control bloat than grass-fed cattle. Do some outside research and use what you have learned in the lab about fermentation to explain this phenomenon. Can you think of any human examples where diet can affect digestive processes involving fermentation? 108

109 Cell Cycle and Mitosis Objectives: 109 Lab 8: The Structure of DNA and DNA Replication 1. Extract DNA from tissue provided and be able to explain how each of the extraction steps relates to the biochemistry of DNA. 2. Be able to describe the basic structure of DNA. 3. Become familiar with the history of the discovery of the structure of DNA. 4. Explain the basic process of transcription. 5. Explain the basic process of translation. Be able to use the genetic code to predict an amino acid sequence when given the DNA template or the mrna. 6. Describe different types of mutations (e.g. base insertion) and discuss briefly the possible effect on the organism. Vocabulary: genes, chromosomes, DNA (deoxyribonucleic acid), pathogenic, phage, nucleotides, deoxyribose, phosphate, nitrogenous base, purine, pyrimidine, adenine, guanine, cytosine, thymine, Chargaff s rule, complementary, replication, mrna, uracil, codon, transcript, RNA polymerase, ribosomes, translation, gene, polypeptide, interphase, cell cycle, semiconservative, DNA polymerase, mutation, base substitutions, base insertions, base deletions, allele, redundancy, genetic code, transcription, frameshift, point mutations (challenge option: restriction enzyme, gel electrophoresis) Microworlds Opportunity: Specimens available in prepared slide set and cultures available in lab may be used as time permits. Your instructor will provide further directions. Introduction: Gregor Mendel s classic experiments in the 1860 s with the common garden pea lead to an understanding of some of the classic principles of genetic inheritance. He described the units of inheritance as factors, which are passed from one generation to the next. His work was largely ignored until the early 1900 s. As interest in the mechanism of inheritance picked up, Mendel s inheritance factors were named genes (in 1909 or so). Evidence for their location on chromosomes began to accumulate, and was clearly demonstrated by the careful work of Thomas Hunt Morgan, working with fruit flies in the 1910 s and 1920 s. Morgan s work suggested specific chromosomal locations for a variety of fruit fly genes. It was known that chromosomes were made up of a mixture of protein and DNA (deoxyribonucleic acid), but it was not known which of those two major types of macromolecules the genetic material was. In fact, many scientists believed that the chemical composition of DNA was far too simple to account for the large number of traits in organisms, and thought that proteins, with their greater complexity, were more likely to carry the genetic information. In 1928, Fredrick Griffiths found that non-pathogenic bacteria could be transformed, becoming pathogenic after being mixed with the heat-killed cells from a truly pathogenic strain

110 Cell Cycle and Mitosis (a pathogen is a disease-causing organism). It wasn t until 1944 that Avery, McCarty and McLeod were able to show that Griffiths transforming principle was DNA. This was brilliantly supported by the T2 phage virus experiment of Hershey and Chase, in which the protein and DNA components of the phage were separately tagged and followed as the virus reproduced inside bacteria. The tagged DNA was found inside the bacterial cells. This demonstrated that DNA, not protein, was the genetic material. Thus, the scientific community of the late 1940 s and early 1950 s knew that DNA was vitally important to understand life and inheritance, but they still did not understand its structure or how it worked to pass on genetic information. Based on the work of Chargaff and others, here is what was known about the structure of DNA by the early 1950 s: 1. DNA is made of subunits called nucleotides. 2. A DNA nucleotide is composed of a sugar (deoxyribose), a negatively charged phosphate and one of 4 possible nitrogenous bases: adenine, thymine, guanine or cytosine. 3. Bases can be of two types: purines (double ringed base structure) or pyrimidines (single ring structure). 4. The purine bases are adenine and guanine. 5. The pyrimidine bases are cytosine and thymine. 6. In the DNA of any organism the number of purines equals the number of pyrimidines. 7. The number of adenines is equal to the number of thymines, and the number of guanines is equal to the number of cytosines. This is Chargaff s rule. James Watson and Francis Crick (1953) published a landmark paper in the journal Nature describing a model for the structure of DNA. Their model was in part based on the experimental data of scientist Rosalind Franklin. Watson, Crick, and Maurice Wilkins were later to receive the Nobel Prize for their work on the structure of DNA. Despite her important contribution to the discovery of the structure of DNA, Franklin did not receive the Nobel Prize partly because she had died of cancer many years before it was awarded. Nucleic acids are composed of nucleotides. On each strand, the phosphate of one nucleotide is covalently linked to the sugar of another, forming a long sugar-phosphate-sugarphosphate backbone. The nitrogen-containing bases, abbreviated as G, A, C and T, stick out. DNA is a double stranded molecule: two strands are arranged so that the bases of one strand can hydrogen-bond with complementary bases on the other strand. As and Ts can hydrogenbond to each other, as can Gs and Cs, but A will not hydrogen-bond with G or C, because the atoms are not in the right place. If you think of the DNA molecule as a twisted ladder, then the rungs are the A-T and the G-C base pairs joined by hydrogen bonds and the sides of the ladder are made of alternating sugar and phosphate groups joined by covalent bonds. Since a base in one strand can only pair with its complementary base from the other strand, if the bases in one strand are known, the bases in the other strand are also known. One of the most famous understatements in science is the line at the end of Watson and Crick s (1953) paper that says, It has not escaped our notice that the specific pairing we have 110

111 Cell Cycle and Mitosis postulated immediately suggests a possible copying mechanism for the genetic material. Accurate DNA replication insures that identical genetic material is in every cell in an organism (and is discussed further below). Now that the structure of DNA was known and the mechanism for copying proposed, the next challenge was to understand how the base sequence of DNA was able to direct the synthesis of proteins. In other words, how does the base sequence of DNA encode genetic information? This puzzle was solved in the 1960s. DNA is a template for mrna (messenger ribonucleic acid). RNA is also a polymer of nucleotides with a sugar-phosphate backbone and the bases dangling off. However, RNA nucleotides have some important differences compared to DNA. RNA nucleotides contain ribose instead of deoxyribose. The nitrogenous bases that can be found in RNA nucleotides include cytosine, adenine and guanine, but NOT thymine. Thymine is replaced by uracil. Uracil is a pyrimidine and complementary to adenine, and follows the rules of complementary base pairing (Quick exam tip: if you are ever asked a question about which sequence of RNA is the right one for a given DNA sequence, never pick the sequence with a T in it). The order of bases in mrna provides directly specifies the order of amino acids in a polypeptide or protein, based on the genetic code. Three adjacent mrna bases make up a codon. This is the code or the key giving the instructions for a specific amino acid. A cell uses the information encoded in the DNA sequence of bases to manufacture proteins. The process of transcription allows RNA polymerase to copy the DNA gene into an RNA molecule that is complementary to one strand, the template strand, of the DNA double helix. In eukaryotes, the resulting mrna is edited and leaves the nucleus of the cell. In the cytoplasm, it hooks up with the ribosomes and is translated from nucleic acid language (groups of three nucleotide codons) to protein language (amino acids). In other words, the mrna has the information to determine which amino acids are brought together and the order in which they are joined to form a polypeptide. The relationship among these molecules had often expressed as DNA to RNA to protein, and has been called The Central Dogma of biology. Understand that in both transcription and translation, it is not a literal conversion of one molecule into another, but information in one molecule being used as the directions or code to make another molecule. The term gene, which was originally introduced to refer to Mendel s inheritance factors, is now understood to mean the coding sequence for a specific protein. The proteins are the molecules that determine traits (e.g. brown hair or red hair, green eyes or blue eyes, etc.). Finally, we return to consider how DNA is copied so that each new cell, and each generation, has a complete set of genetic information. Each DNA molecule replicates itself during interphase of the cell cycle, which maintains identical genetic information in new cells that form. When DNA replicates, the two strands separate, and enzymes, such as DNA polymerase, use each strand as a template to assemble new nucleotides into a complementary strand. Since each new DNA molecule contains one old strand and one new strand, replication is said to be semi-conservative. Occasionally, errors are made during replication. Such an error is a mutation. Point mutations are changes in a single nucleotide base pair. If a 111

112 Cell Cycle and Mitosis nucleotide with one type of nitrogenous base is switched to another, it is called a base substitution. If an extra nucleotide is slipped into the sequence, it is called a base insertion. Removal of a single nucleotide from the sequence would be a base deletion. If these changes occur in a region of the DNA where there is a coding sequence or a gene, then a new allele or new versions of the gene may result. Sometimes the change does not affect the protein product and sometimes the change is catastrophic for the organism. Very rarely the change actually results in an improved protein product. Redundancy of the genetic code protects against the effects of mutations. Redundancy means that there are some amino acids that are coded for by more than one codon just like different words in the English language that have the same meaning. Although it is impossible to see the nucleotides in a DNA molecule with the naked eye, it is possible to see whole DNA molecules isolated from cells. The procedure for isolation of DNA from tissues typically involves some very simple steps and very basic chemistry. First, the tissue is crushed or ground to physically break any connective tissue or cell walls that may be present. Second, a solution containing detergent and NaCl is used to break down the phospholipid membranes and the internal membranes of the cell. Recall that detergents and soaps have chemical properties similar to those of phospholipids soaps and detergents are amphipathic, and will interact with both the polar and non-polar regions of the phospholipid bilayer, disrupting them and releasing the contents of the cell and the nucleus. The sodium chloride dissociates in solution, and the ions disrupt protein and other structures causing them to precipitate out of solution. The liquid portion of the lysis buffer/tissue sample is then separated from the solid portion by filtration. The filtrate that is produced contains DNA in soluble form the molecules can not yet be seen. To precipitate the DNA from solution, ethanol or another alcohol is used to make the DNA insoluble. Since DNA is polar, the ethanol, which is less polar than water, along with sodium ions in solution, disrupts the interaction of DNA with the water molecules in solution, causing it to precipitate out of solution. This mass of DNA can be collected with a glass rod for observation, or can be further purified for other procedures. 112

113 Cell Cycle and Mitosis Exercise 1: DNA Extraction Equipment Needed Per Group: Test tube rack Funnel Coffee filter Small glass beaker: put on ice you will put the cold ethanol from the freezer in this beaker, but do not fill until you are ready to use it Ice cold 70% ethanol: in freezer; do not take until you are ready to use 50 ml graduated cylinder Test tube Re-sealable plastic bag 1 whole strawberry Lysis buffer Pasteur pipette or glass rod Methylene blue Gloves and safety goggles Procedure: 1. Gather all the materials you will need (except ice cold ethanol) including gloves and goggles, and read through all of this procedure before beginning. Place the small beaker on ice. 2. Remove the leaves from the top of strawberry and place fruit in re-sealable plastic bag. 3. Seal sandwich bag and squash the fruit with your palm. Squash as thoroughly as possible without breaking the bag. 4. Add 10 ml of lysis buffer to the bag with the pulverized fruit. Continue to squash mixture for an additional two minutes. 5. Place a filter in the funnel and suspend funnel over glass beaker. If you do not have a funnel, fold the filter paper into a cone and suspend in a small glass beaker. 6. Pour the contents of the plastic bag into the filter and set aside for 10 minutes. 7. Discard filter and contents. 8. Pour the filtrate into a test tube (estimate the volume) 9. You will need approximately twice the volume of ice-cold 70% ethanol as your filtrate. 10. Designate someone in your group to take the beaker that has been on ice and go to the freezer to get the amount of ethanol needed by your group. 113

114 Cell Cycle and Mitosis 11. From the ethanol (keep on ice as much as possible), use a graduated cylinder to measure out a volume of ethanol approximately equal to the amount of strawberry filtrate in your test tube. 12. SLOWLY pour the ethanol down the side of the test tube with the strawberry filtrate. The DNA will start precipitating out in the alcohol almost immediately, but you will want to gently put your test tube in the rack and wait a few minutes to get the maximum effect. DO NOT MIX THE CONTENTS. You want the ethanol to remain a distinct layer on the top of the filtrate. 13. OPTIONAL: Use the end of the Pasteur pipette (or glass rod) to carefully spool the precipitated DNA out of the ethanol. Put some DNA on a clean glass slide, add a drop of methylene blue, and place a coverslip on top. You can then view the DNA through a microscope. 14. CLEAN up all material used as directed in lab. DO NOT put Pasteur pipettes in the garbage they should go in the broken glass container. Glass rods can be washed and returned to the equipment tray. Literature Cited: Peters, P The Structure of the DNA, Access the National Health Museum Accessed September 13, Watson, J.D., and F.H.C. Crick Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 171:

115 Cell Cycle and Mitosis Exercise 2: DNA Transcription, Translation and Mutations Be able to answer questions based on this exercise for the quiz. 1. Add the missing bases to this strand of DNA. A T C G T C T T G 2. At the left is a hypothetical segment of DNA. During replication, for the purpose of cell division, the strands will separate and DNA polymerase will read each strand and build new, complementary strands of DNA. Add the missing bases in the newly synthesized strands. A C G G T T A C G G T T G C C A DNA Replication A T G C C A 3. The figure below shows a portion of a DNA molecule whose strands have been separated for synthesis of mrna. Fill in the bases of the DNA template strand, and then fill in the bases of the newly forming mrna strand. A T G G C G A C A T A T newly forming mrna DNA template strand 115

116 Cell Cycle and Mitosis 4. It is common for scientists to work backwards to find a gene of interest. They start by determining the sequence of amino acids in a protein that interests them. From the amino acid sequence, they can determine possible mrna codons and then determine the possible DNA sequence for the gene of interest. They can then construct the gene and express it in a plant or microbe so that they can produce larger quantities of the protein. Indicate one possible mrna codon and the DNA base triplet for each amino acid. Amino Acid mrna codon DNA base triplet Lysine Serine Arginine Valine 5. Consider the following hypothetical gene. Transcribe the DNA to form mrna codons, and then give the sequence of amino acids that forms the polypeptide during translation. mrna codons Amino acid sequence T-A-C-T-T-A-C-C-G-T-C-A-A-T-C 6. For the following mutations in the gene above, show the effect of the mutation by indicating the mrna codons and the amino acid sequence. a. base substitution T-A-C-T-T-A-C-C-G-T-C-G-A-T-C mrna codons Amino acid sequence b. base insertion T-A-C-T-T-A-T-C-C-G-T-C-A-A-T-C mrna codons Amino acid sequence (note how a base insertion can cause a frameshift in the codons) 116

117 Cell Cycle and Mitosis 6 (continued) c. base deletion T-A-C-T-T-A-C-C-G- -C-A-A-T-C mrna codons Amino acid sequence (note how a base deletion can also cause a frameshift in the codons) a, b and c are all examples of point mutations, i.e. changes in only one base, but changes can occur in the DNA on a larger scale. For example: d. triplet addition T-A-C-T-T-A-C-C-G-T-C-A-G-C-A-A-T-C mrna codons Amino acid sequence (note: Huntington s Disease is caused by having 30 or more CAG repeats) 7. Examine the different amino acid sequences (6a, b, c and d above) and compare them to the first sequence given in #5. In general, what types of mutations change the amino acid sequence the most? Be able to explain why this could lead to a protein product that was not functional. A U C G 117 AAA - lys AAU - asn AAC asn AAG - lys UAA - stop UAU - tyr UAC - tyr UAG - stop CAA - gln CAU - his CAC - his CAG - gln GAA - glu GAU - asp GAC - asp GAG - glu A U C G AUA - ile ACA - thr AGA - arg AUU - ile ACU - thr AGU - ser AUC - ile ACC thr AGC - ser AUG start (met) ACG - thr AGG - arg UUA - leu UUU - phe UUC - phe UUG - leu CUA - leu CUU - leu CUC - leu CUG - leu GUA - val GUU - val GUC - val GUG - val UCA ser UCU - ser UCC - ser UCG - ser CCA - pro CCU - pro CCC - pro CCG - pro GCA - ala GCU - ala GCC - ala GCG - ala Table 1. Codon table UGA - stop UGU - cys UGC - cys UGG - trp CGA - arg CGU - arg CGC - arg CGG - arg GGA - gly GGU - gly GGC - gly GGG gly A U C G A U C G A U C G A U C G

118 Cell Cycle and Mitosis DNA Study Guide Questions Be able to answer for a quiz. 1. What is the purpose of the detergent? Hint: think about where the DNA is within the cell. What would you have to do to get the DNA out of the cell? What other macromolecules might get in the way? 2. What is the purpose of the salt? 3. Other protocols add enzymes such as those found in meat tenderizer or fresh papaya juice. What would the enzyme accomplish? 4. Other protocols heat the filtrate up to 50 or 60 C. What would the heat accomplish? (Note: DNA does not denature until 80 C) 5. Why does the DNA precipitate in the ethanol? 6. Would any tissue work just as well for this experiment? 7. Is the precipitate pure DNA? 8. Is the nucleus the only place that DNA is found inside the cell? You may have to check your textbook for this answer. 9. The DNA extraction carried out in lab today could has three possible outcomes: a. A large quantity of stringy DNA is recovered b. DNA is recovered but it is fluffy or in bits c. No DNA is recovered. Try to explain what might have occurred for outcomes (b) and (c). Challenge Question: One of the most famous understatements in science is the line at the end of Watson and Crick s (1953) paper that says, It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Can you explain what they meant? Challenge Question: Evolution is a process whereby the genetic composition of a population changes over time. It is said that mutation is the raw material for evolution. Can you explain this statement based on your understanding of the relationship between the sequence of bases in a gene (DNA) and the protein product? 118

119 Cell Cycle and Mitosis Objectives: 119 Lab 9a: Cell Cycle and Mitosis 1. State the purpose of mitosis. 2. Describe the events occurring in each of the following phases of the cell cycle -G 1, S, G 2, G 0, prophase, metaphase, anaphase, telophase, and cytokinesis. 3. Identify the phases of mitosis in plant and animal cells. 4. Explain how mitosis differs in plant and animal cells. Vocabulary: mitosis, meiosis, binary fission, cell cycle, interphase, cytokinesis, chromosomes, chromatin, sister chromatids, centromere, centrioles, prophase, metaphase, anaphase, telophase, mitotic spindle, cleavage furrow, cell plate, somatic cells diploid, haploid, homologous chromosomes, blastula, Allium. Microworlds Opportunity: Your instructor may give you more specific instructions. Introduction: Reproduction of cells is the result of cellular division. Two common mechanisms of cell division among eukaryotes are mitosis and meiosis. Mitosis is a process of nuclear division resulting in two genetically identical daughter cells, which are genetically identical to their parent cell. In this process, the chromosome number of the parent cell is maintained in the resulting daughter cells. In asexually reproducing species, mitosis functions in the creation of new organisms that are genetically identical to their parents. Mitosis is also the basis for growth, repair of damaged tissues, and replacement of older cells in multicellular organisms. The process of meiosis accounts for the production of cells for sexual reproduction. The daughter cells produced by meiosis each have half the number of chromosomes of the original parent cell. Mitosis, which we will be studying today, is one part of the cell cycle, a sequence of events that include the process of cell growth, preparation for division including the replication of DNA, and the formation of new daughter cells (Figure 1). The steps of the cell cycle are predictable and can be separated into distinct stages: interphase and mitosis. During interphase, the genetic material is surrounded by a nuclear envelope and chromosomes are uncoiled and decondensed. The uncoiled DNA and associated proteins are called chromatin. Interphase is divided into three steps: G 1, S, and G 2. During the G 1 phase, cells are carrying out their normal function according to cell type, growing larger, and doubling the material in the cytoplasm. During the S phase, DNA chromosomes are replicated. The two DNA molecules formed when a chromosome is replicated are attached to each other at a point called the centromere and are referred to as sister chromatids. Each replicated chromosome consists of two sister chromatids, joined together. During the G 2 phase, cells continue to grow, and proteins that are necessary for cell division are synthesized.

120 Cell Cycle and Mitosis When cells are not preparing for mitosis, they can enter a phase called G 0. During this phase the cell is metabolically active and performs its normal functions. When trying to identify cells in interphase microscopically, look for a nucleus that contains one or more dark staining nucleoli (site of ribosomal RNA synthesis) and a lack of distinct chromosomes. G 1 (G 0 ) M Prophase Metaphase Anaphase Telophase Cytokinesis S Interphase = G 1 +S+G 2 G Figure 1. The cell cycle. Once interphase has been completed, the process of mitosis can begin. Mitosis is the division of the nucleus and its material. The events occurring during mitosis can be separated into a series of phases: prophase, metaphase, anaphase, and telophase. During prophase, the DNA coils and winds, becoming condensed and visible chromosomes. Nucleoli are no longer visible in the nucleus. The nuclear envelope breaks down and is no longer visible. Centrosomes, the microtubules organizing centers of the cell, migrate to opposite poles. In animal cells, the centrosomes each contain two centrioles, the function of which is a current topic of research. The centrosomes of plant cells lack centrioles. The mitotic spindle forms at this time and is composed of protein fibers, which will attach to the centromeres of the chromosomes and move the chromosomes during the next three phases. During metaphase, the mitotic spindle is fully formed and the chromosomes have been arranged along an imaginary line bisecting the cell. The chromosomes are positioned in such a way that one sister chromatid is on one side of the line and the other sister chromatid is on the other side of the line. During anaphase, the sister chromatids are separated and moved to opposing poles by the mitotic spindle. Once the two sister chromatids are separated, each is referred to as a single chromosome. During telophase, the nuclear envelope is reformed, chromosomes begin to decondense, and the mitotic spindle is broken down. The final phase of the cell cycle is cytokinesis. Biologists differ as to whether cytokinesis is a phase of mitosis or a stage of the cell cycle that follows mitosis. You may notice this

121 Cell Cycle and Mitosis difference when referring to different biology textbooks. While mitosis describes the division of the nucleus and its contents, cytokinesis describes the division of the cytoplasm and its contents. In animal cells, cytokinesis occurs when contractile proteins cause the cell membrane to pinch in, creating a cleavage furrow between the two new cells. In plant cells, a cell plate is formed, establishing a new cell wall and cell membrane between the two new cells. In both cases, two new daughter cells are formed, each of which is genetically identical to the parent cell. It should be pointed out that cytokinesis does not always follow mitosis how many nuclei would be in cells undergoing mitosis but not cytokinesis? Exercise 1a: Modeling the Cell Cycle with Clay (Modified with permission by Jennifer Schramm, Ph.D.) You will model the phases of the cell cycle using clay to represent chromosomes. Somatic (body) cells are diploid (2N); they contain two copies of every chromosome. Sex cells (gametes), on the other hand, are haploid (1N) and contain only one copy of each type of chromosome. In somatic cells, one copy of each chromosome comes from your mother (maternal) and the other copy comes from your father (paternal). These pairs of like chromosomes are called homologous chromosomes. Human cells have 46 chromosomes (23 pairs of homologous chromosomes). You will model the cell cycle with only 6 total chromosomes (3 homologous pairs). During the S phase of interphase, each member of a homologous pair undergoes replication. The two copies formed from the original chromosome remain joined together at a point called the centromere until they are pulled apart during the anaphase stage of mitosis. These joined copies are called sister chromatids. Note that the members of each homologous pair have the same shape, centromere position and genes. Equipment Needed: Modeling clay (3 colors) 2 pieces of string Procedure: 1. Gather the required materials, and read through this procedure before beginning. 2. Using one color of modeling clay, make three snakes of different lengths. Each snake represents a single maternal chromosome. 3. Using another color of modeling clay, make three snakes that match the first set in length. Each of these chromosomes represents the paternal homologues. 4. Using the remaining color of modeling clay, make 6 small balls; representing centromeres. Attach one centromere to the center of each chromosome. You are now ready to begin modeling. 121

122 Cell Cycle and Mitosis 5. Modeling: a. Begin with your cell in G 1. Randomly arrange your unduplicated chromosomes in the center of your cell (your desktop). Place a piece of string around the chromosomes to represent the nuclear envelope. During this time there are no changes to the chromosomes and the cell is growing and performing its normal functions. Remember that in a real cell, interphase chromosomes are not visible, even with the aid of a compound microscope. b. To model the S phase of interphase, you will need to replicate your chromosomes. Add a second clay snake to each of your chromosomes to make an X-shaped structure (be sure your color matches the original chromosome). This X-shaped structure is still called a chromosome, but this duplicated chromosome contains two identical sister chromatids. Each sister chromatid contains its own centromere, but when they are attached the centromeres overlap, appearing as one. c. To model the G 2 phase of interphase, do nothing. Although there are changes occurring in the cell during this phase, there are no changes to the chromosomes. d. To model prophase of mitosis, remove the string representing the nuclear envelope. e. To model metaphase of mitosis, arrange the chromosomes along an imaginary line so that the sister chromatids of individual chromosomes are separated by the imaginary line. f. To model anaphase of mitosis, separate the sister chromatids of each of your chromosomes and begin to move them apart, toward opposite ends of the cell. g. To model telophase of mitosis, continue moving the chromosomes apart and place a piece of string around each set of chromosomes to represent the new nuclear envelopes. h. To model cytokinesis (which often overlaps telophase in a cell); arrange the chromosomes randomly in each of two nuclei to represent two different cells. 122

123 Cell Cycle and Mitosis Exercise 1b: Modeling the Cell Cycle with Pop Beads Equipment Needed: Red and yellow pop beads 8 white magnets 4 centrioles (clear plastic tubes) Procedure: 1. Gather the required materials, and read through this procedure before beginning. 2. Using the red pop beads, make two strands of different lengths use the white magnets as centromeres and the beads as arms. These are the maternal chromosomes. 3. Using the yellow pop beads make two strands of different lengths use the white magnets as centromeres and the beads as arms. These are the paternal chromosomes. 4. Modeling a. Begin with your cell in G 1. Randomly arrange your unduplicated chromosomes in the center of your cell (your desktop). Place a piece of string around the chromosomes to represent the nuclear envelope. During this time there are no changes to the chromosomes and the cell is growing and performing its normal functions. Remember that in a real cell, interphase chromosomes are not visible, even with the aid of a compound microscope. b. To model the S phase of interphase, you will need to replicate your chromosomes. Using the remaining pop beads and magnets, make copies of the existing chromosomes. Be sure to attach your sister chromatids at the centromere (the magnets will stick together). c. To model the G 2 phase of interphase, do nothing. Although there are changes occurring in the cell during this phase, there are no changes to the chromosomes. d. To model prophase of mitosis, remove the string representing the nuclear envelope. e. To model metaphase of mitosis, arrange the chromosomes along an imaginary line so that the centromeres are along the line and the chromatids are above and below the line (equator of the cell). f. To model anaphase of mitosis, separate the sister chromatids of each of your chromosomes and begin to move them apart, toward opposite ends of the cell. g. To model telophase of mitosis, continue moving the chromosomes apart and place a piece of string around each set of chromosomes to represent the new nuclear envelopes. h. To model cytokinesis (which often overlaps telophase in a cell); arrange the chromosomes into each of two nuclei to represent two different cells. 123

124 Cell Cycle and Mitosis Exercise 2: Identifying Mitotic Phases in Plant Cells You will use slides of Allium (onion) root tips to identify cell cycle and mitotic phases in plant cells. The tips of plant roots are areas of rapid growth so there are many cells here undergoing mitosis. Please identify each of the following: interphase, prophase, metaphase, anaphase, telophase, and cytokinesis. In plant cells, cytokinesis is characterized by the formation of a cell plate across the equator of the cell, dividing the cell in two. Equipment Needed: Microscope Slide of Allium root tip longitudinal sections Procedure: 1. Gather the required equipment; read through this procedure before beginning. 2. Prior to setting the slide in the scope, look at it without magnification. Most of our root tip slides have several longitudinal-sections of root tip prepared. Each cross-section contains many cells; not all cells with be in the same phase. You may be able to identify all phases within one longitudinal-section, or you may need to observe several sections. 3. Place the slide on the stage of the microscope; locate one cross-section of root tip. Find the very tip of the root. This is the root cap, a group of cells that protect the newly formed tissue. Just above the root tip is the region of rapid cell division. The chromosomes are stained a dark color. 4. Identify cells in interphase, prophase, metaphase, anaphase, telophase, and cytokinesis, and draw a sketch of what a cell in each phase looked like in the space provided. Onion root tip Interphase Onion root tip Prophase Onion root tip Metaphase Onion root tip Anaphase Onion root tip Telophase 124

125 Cell Cycle and Mitosis Exercise 3: Identifying Mitotic Phases in Animal Cells You will use slides of fish blastula to identify cell cycle and mitotic phases in animal cells. Whitefish is the common name of any fish in the genus Coregonus (N. American freshwater fish). The term blastula refers to an embryonic developmental stage. The fish embryos are small and contain cells that are rapidly dividing. This is useful for studying mitosis. Please identify each of the following: interphase, prophase, metaphase, anaphase, telophase, and cytokinesis. Remember that in animal cells, cytokinesis is characterized by the formation of a cleavage furrow containing contractile proteins. As these proteins contract, the cleavage furrow tightens and pinches the cell into two new daughter cells. Equipment Needed: Microscope Slide of fish blastula cross-sections Procedure: 1. Gather the required equipment, and read through this procedure before beginning. 2. Prior to setting the slide in the scope, look at it without magnification. Most of our fish blastula slides include more than one cross-section of a blastula. Each cross-section contains many cells; not all cells will be in the same phase. You may be able to identify all phases within one cross-section, or you may need to observe more than one cross-section. 3. Place the slide in the microscope, and locate one cross-section of blastula. 4. Identify cells in interphase, prophase, metaphase, anaphase, telophase, and cytokinesis, and draw a sketch of what a cell in each phase looked like in the space provided. Whitefish blastula Interphase Whitefish blastula Prophase Whitefish blastula Metaphase Whitefish blastula Anaphase Whitefish blastula Telophase 125

126 Cell Cycle and Mitosis Exercise 4: Estimating the Time Spent in the Phases of Mitosis Now that you are able to identify cells in the different phases of the cell cycle, you can use this skill to develop an understanding of how long this process takes and how long a cell is in each of the phases. You can use either the whitefish blastula slide or the onion root tip slide. Equipment Needed: Microscope Slide of Allium root tip longitudinal sections or slide of whitefish blastula sections Procedure: 1. Gather a microscope and one of the slides, and read through this procedure before beginning. 2. Identify the phases of the cell cycle for each of 25 randomly-chosen cells. Record your data in Table 1 below. It is important that you choose cells randomly so that you do not bias your data. 3. Trade data with 3 other people so that you have a total of 100 cells identified. Table 1. Number of cells in each phase of the cell cycle. Interphase Prophase Metaphase Anaphase Telophase Total 1 st 25 cells 25 2 nd 25 cells 25 3 rd 25 cells 25 4 th 25 cells 25 TOTAL Calculate the percentage of time spent in each phase by counting the total number of cells in each phase (total in interphase, total in prophase, and so on) and dividing each by the total number of cells identified (which should be 100!). Enter your results in Table 2 below. 5. Multiply the percentage of time in each phase by the total time of the cell cycle (in an onion root tip, the cell cycle takes about 12 hours, so use that figure). Enter your results in Table 2 below. Table 2. Estimate of time spent in each phase of the cell cycle. Interphase Prophase Metaphase Anaphase Telophase Total % of cells 100% Time estimate 12 hours 126

127 Cell Cycle and Mitosis Mitosis Study Guide Questions Be able to answer for a quiz. 1. Be able to recognize the correct sequence of the stages of cell division. Be able to recognize the major events of each stage. 2. Be able to recognize on microscope slides any stage of cell division in either animal or plant on microscope slides. 3. Be able to recognize the following structures on microscope slides: cell wall, cell plate, cleavage furrow, mitotic spindle, nucleus, chromatin, chromosomes 4. What is the distinction made between mitosis and cytokinesis? 5. How does cytokinesis differ between animal and plant cells? 6. Do most plants have centrioles? 7. What is a blastula? 8. What is Allium? 9. Why are root tips often used to observe mitosis in plants? 10. What is meant by diploid? 11. What is meant by haploid? 12. What is the difference between a pair of homologous chromosomes and sister chromatids? 13. What are somatic cells? 14. During what part of the cell cycle does DNA synthesis occur (be as specific as possible)? 15. During which phases of the cell cycle (be as specific as possible) are the chromosomes in the replicated form (containing two sister chromatids)? Challenge Question: PET (positron emission tomography) is often used for early detection of cancerous tumors. In this case, radiolabeled glucose is administered to the patient and its distribution in the body is monitored by the PET. Can you explain why tracking the distribution of glucose in the body might help detect a tumor? 127

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129 Meiosis Lab 9b: Meiosis Objectives: 1. Describe the function of meiosis. 2. Describe the events occurring in each phase of meiosis. 3. Describe independent assortment. 4. Explain how independent assortment is related to genetic variation. 5. Describe the process of crossing-over. 6. Explain how crossing is related to genetic variation. Vocabulary: diploid (2N), homologous pairs, haploid (1N), fertilization, gametogenesis, sister chromatid, centromere, allele, meiosis I, prophase I, crossing-over, synapsis, tetrad, chiasmata, genetic recombination, independent assortment, metaphase I, anaphase I, telophase I, meiosis II, prophase II, metaphase II, anaphase II, telophase II, Introduction: Sexual reproduction provides an evolutionary advantage by allowing for greater genetic diversity within the population. Sexual reproduction results in genetically unique offspring, compared to asexual reproduction, which usually produces offspring that are genetically identical to their parents. Human somatic (body) cells are diploid (2N). This means that they have two of each type of chromosome. Human cells have 23 different types of chromosomes. A normal somatic cell would have a total of 46 chromosomes: two of each type or 23 homologous pairs. One of the chromosomes is maternally inherited and the other is paternally inherited. In humans, diploid cells cannot come together to form a new organism. For a new organism to form, special haploid (1N) gamete cells must be made. These haploid cells contain half the genetic material of a somatic cell. During sexual reproduction, the gametes most animals produce are sperm and eggs. When the sperm and the egg join and combine their nuclear chromosomes (fertilization), the result is a cell that is once again diploid. The mechanism for producing sperm and egg is called gametogenesis. The process of gametogenesis occurs with one DNA replication and two cellular divisions. The result is four cells, each with ½ the original chromosome number. Each new cell contains one individual chromosome from each homologous pair and is considered haploid or 1N. We will discuss gametogenesis in three main steps: DNA replication, meiosis I, and meiosis II. DNA replication in preparation for meiosis occurs during the S phase of interphase. The DNA replication occurring here is the same as that occurring prior to mitosis. It results in each individual chromosome consisting of two identical sister chromatids attached at the centromere. 129

130 Meiosis Chromosome number is reduced to the haploid state during the first cellular division of meiosis, meiosis I. Meiosis I occurs in four phases: prophase I, metaphase I, anaphase I, and telophase I. During prophase I, DNA coils and winds, becoming condensed chromosomes. The nuclear envelope breaks down, nucleoli are usually absent, and the mitotic spindle reforms. Also during prophase I, exchange of genetic material between non-sister chromatids of homologous chromosome pairs occurs. This process, resulting in genetic recombination, is called crossing-over. During crossing-over, homologous pairs of chromosomes come together in a process called synapsis. Synapsis results in the formation of tetrads, a structure consisting of two homologous chromosomes, each consisting of two sister chromatids. While in the tetrads, the non-sister chromatids of the homologous pair form chiasmata, locations where the chromatids physically cross each other. This allows the non-sister chromatids to exchange portions of the chromatids, resulting in new combinations of alleles (genetic recombination). At this point, the sister chromatids in each chromosome are no longer identical. Although they contain the same genes, they now have a new combination of alleles for those genes. During metaphase I, the tetrads are arranged along the midline of the cell. Homologous pairs are aligned so that each daughter cell will receive a single chromosome of that pair. Each homologous pair aligns independently of how the other pairs are arranged. This is independent assortment. During anaphase I, the homologous pairs of chromosomes are pulled apart. Although the homologous pairs are separated, each chromosome consists of a pair of sister chromatids. During telophase I, the chromosomes reach the poles. At the end of meiosis I, two haploid cells have formed. Each cell contains one of the chromosomes from each homologous pair in the parent cell. Although chromosome number is reduced in meiosis I, each chromosome still contains two sister chromatids. To produce functional gametes, these sister chromatids must be separated into two different cells. This occurs during the process of meiosis II. Meiosis II can be described in four phases: prophase II, metaphase II, anaphase II, and telophase II. During prophase II, a new spindle is produced, which attaches to the chromosomes and moves them toward the midline of the cell. During metaphase II, the chromosomes line up along the midline of the cell. During anaphase II, the sister chromatids are separated and they move toward opposing poles. Each sister chromatid is now called a chromosome. During telophase II, the chromosomes reach the poles and a nuclear envelope is formed at each pole. The process of cytokinesis occurs resulting in 2 haploid daughter cells. Recall that this process occurs for each of the daughter cells produced during meiosis I. Therefore, there are four total cells produced at the end of meiosis II for each cell that began meiosis I. Each of the four daughter cells produced at the end of meiosis is genetically unique due to crossing-over and independent assortment. Recall that one advantage of sexual reproduction is genetic diversity. Sources of genetic diversity include independent assortment of chromosomes, crossing-over, random fertilization and mutation. How the homologous pairs line up at the cell midline at metaphase I is random, 130

131 Meiosis therefore a single gamete inherits a chromosomes that are from all grandparents. Crossing-over that occurs during prophase I contributes to genetic diversity by allowing for a greater number of possible allele combinations in the gametes produced by an individual. A single female will produce many eggs, each of which is genetically unique. The millions of sperm produced by a male will also represent many different possible genetic combinations. When the egg and sperm meet and fertilization occurs to create a new organism (zygote), it is not possible to predict which egg will be fertilized by which sperm. Since fertilization is random, many different combinations are possible. Although not part of the process of meiosis, mutations are also an important source of genetic diversity. Today, you will model meiosis with modeling clay. Pop bead sets are also available in the lab if you would prefer to use those. Exercise 1a: Modeling Meiosis with Modeling Clay (Modified with permission by Jennifer Schramm, Ph.D.) Equipment Needed: Modeling clay (3 colors) Procedure: 1. Gather the required materials, and read through this procedure before beginning. 2. Using one color of modeling clay, make three snakes of different lengths. Each snake represents a single maternal chromosome. 3. Using another color of modeling clay, make three snakes that match the first set in length. Each of these chromosomes represents the paternal homologues. 4. Using the remaining color of modeling clay, make 6 small balls (representing centromeres). Attach one centromere to the center of each chromosome. 5. Modeling: 131 a. To model the DNA replication, which occurs prior to meiosis, add a second clay snake to each of your chromosomes to make an X-shaped structure (be sure your color matches the original chromosome). This X-shaped structure is still called a chromosome, but this duplicated chromosome contains two identical sister chromatids. Each sister chromatid contains its own centromere, but when they are attached, the centromeres overlap, appearing as one. Are the cells haploid or diploid at the end of this step? b. To model crossing-over, which occurs during prophase I, move the two chromosomes in each homologous pair together to form the tetrad. Remove equal size pieces of one paternal sister chromatid and one maternal sister chromatid. Reattach the maternal piece to the paternal chromosome and the paternal piece to the maternal chromosome.

132 Meiosis c. To model metaphase I of meiosis, arrange the chromosomes along an imaginary line so that the individual chromosomes of homologous pairs are separated by the imaginary line. d. To model anaphase I of meiosis, separate the individual chromosomes of each of the homologous pairs and begin to move them apart, toward opposite ends of the cell. e. To model telophase I of meiosis, continue moving the chromosomes apart. f. To model cytokinesis I, arrange the chromosomes into each of two nuclei to represent two different cells. Are the cells haploid or diploid at the end of this step? g. To model metaphase II of meiosis, arrange the chromosomes, in both cells, along an imaginary line so that the sister chromatids of an individual chromosome either side of the equator of your cell. 132

133 Meiosis h. To model anaphase II of meiosis, separate the sister chromatids of each of the homologous pairs and begin to move them apart, toward the opposite poles of each cell. i. To model telophase II of meiosis, continue moving the sister chromatids, now called chromosomes, apart. j. To model cytokinesis II, arrange the chromosomes into each of four nuclei to represent four different cells. Are the cells haploid or diploid at the end of this step? How many chromosomes are in each daughter cell? Are the daughter cells genetically identical? How is the correct number of chromosomes restored? 133

134 Meiosis Exercise 1b: Modeling Meiosis with Pop Beads Equipment Needed: Red and yellow pop beads 8 white magnets 4 centrioles (clear plastic tubes) Procedure: 1. Gather the required materials, and read through this procedure before beginning. 2. Using the red pop beads, make two strands of different lengths use the white magnets as centromeres and the beads as arms. These are the maternal chromosomes. 3. Using the yellow pop beads, make two chromosomes. Each should be homologous to one of the maternal chromosomes. These are the paternal chromosomes. 4. Modeling a. To model the S phase of interphase, you will need to replicate your chromosomes. Using the remaining pop beads and magnets, make copies of the existing chromosomes. Be sure to attach your sister chromatids at the centromere (the magnets will stick together). b. To model crossing over and prophase I of meiosis, move the two chromosomes in each homologous pair together to form the tetrad. Exchange an equal number of beads between one chromatid of the paternal chromosome with one chromatid of the maternal chromosome you may do this for both homologous pairs. c. To model metaphase I of meiosis, arrange the chromosomes along an imaginary line so that the individual chromosomes of homologous pairs are separated by the imaginary line. d. To model anaphase I of meiosis, separate the individual chromosomes of each of the homologous pairs and begin to move them apart, toward opposite ends of the cell. e. To model telophase I of meiosis, continue moving the chromosomes apart. f. To model cytokinesis I, arrange the chromosomes into each of two nuclei to represent two different cells. Are the cells haploid or diploid at the end of this step? 134

135 Meiosis g. To model metaphase II of meiosis, arrange the chromosomes, in both cells, along an imaginary line so that the sister chromatids of an individual chromosome either side of the equator of your cell. h. To model anaphase II of meiosis, separate the sister chromatids of each of the homologous pairs and begin to move them apart, toward the opposite poles of each cell. i. To model telophase II of meiosis, continue moving the sister chromatids, now called chromosomes, apart. j. To model cytokinesis II, arrange the chromosomes into each of four nuclei to represent four different cells. Are the cells haploid or diploid at the end of this step? How many chromosomes are in each daughter cell? Are the daughter cells genetically identical? How is the correct number of chromosomes restored? 135

136 Meiosis Meiosis Study Guide Questions Be able to answer for a quiz. 1. Fill in the following table to compare and contrast mitosis and meiosis. Mitosis Meiosis Purpose Type of tissue involved Number of cell divisions Crossing-over of non-sister chromatids Number of daughter cells produced Final cells produced are haploid or diploid Genetic composition of daughter cells compared to parent cell 2. At the end of meiosis I, are the cells haploid or diploid? 3. At the end of meiosis II, are the cells haploid or diploid? 4. What structures are being separated in meiosis I: sister chromatids or homologous chromosomes? 5. What structures are being separated in meiosis II: sister chromatids or homologous chromosomes? 6. Explain two events in meiosis that contribute to genetic diversity. Be sure you can identify the phase(s) during which each event occurs. 136

137 Mendelian Genetics Lab 10: Genetics Objectives: 1. Use Punnett squares to predict potential offspring genotypes and phenotypes when provided with parental genotypes and type of inheritance (autosomal dominant, autosomal recessive, codominance, incomplete dominance, X-linked dominant and X-linked recessive). 2. Know how to calculate the probability of individual events and consecutive events. Vocabulary: Gregor Mendel, allele, gene, phenotype, genotype, dominant, recessive, gametes, Law of Segregation, Law of Independent Assortment, Punnett square, genotype, homozygous dominant, homozygous recessive, heterozygous, monogenic, polygenic, complex, autosomal dominant, autosomal recessive, carrier, codominance, ABO blood group, incomplete dominance, hemizygous, X-linked dominant, X-linked recessive, probability Microworlds Opportunity: Your instructor may give you more specific instructions. Introduction: Genetics is the study of how DNA-based information is passed from one generation to the next. As you will learn in lecture, Gregor Mendel pioneered the field of genetics with his studies of inheritance in the common garden pea. Mendel worked meticulously, moving tiny grains of pollen from the flower of a specific plant to the stigma (female receptacle) of a different plant. Over many years and many generations of garden peas, Mendel devised his Theory of Inheritance. This theory can be broken down into five major parts: 1. Genetic variation is the result of the presence of different versions (alleles) of genes. 2. Offspring have two copies of every gene, one from each parent. 3. If the two copies of a gene present in an individual are different, only one version of the gene will be observable in the offspring. The observable traits in an individual that are due to inheritance are referred to as that individual s phenotype. An individual s genetic makeup is called its genotype. This allele is known as the dominant allele and the other allele (which is not evident in the phenotype) is called the recessive allele. 4. During meiosis, the two copies of the gene present in an individual separate into different gametes (this separation is known as the Law of Segregation). 5. When examining more than one gene, each allele of each gene will separate into gametes independent of the how the alleles of other gene(s) separate (known as the Law of Independent Assortment). 137

138 Mendelian Genetics Exercise 1: Introduction to Punnett Squares and Probability (Modified with permission by Jennifer Schramm, Ph.D.) The worksheet below is designed to help you understand the relationship between genetics and meiosis. You will also be introduced to the Punnett Square, a useful tool for completing genetics problems. Remember that the best way to understand genetics is to practice. 1. Draw the labeled chromosomes in each cell below (if you just use letters, it is difficult for your instructor to check your understanding). As you work, answer the questions in the middle of the diagram. The two chromosomes within one cell represent a homologous pair. Each half of one of these chromosomes is a sister chromatid. Paren t 1 P are n t 2 A A a a G erm ce ll s at the be ginnin g o f Pr ophase I of m e iosis H a p l o i d o r d i p l o i d? C h r o m o s o m e s d u p l i c a t e d o r u n d u p l i c a t e d? a a a a P roducts of M eiosis I H a p l o i d o r d i p l o i d? C h r o m o s o m e s d u p l i c a t e d o r u n d u p l i c a t e d? P roduc Products of M eios of is I Meiosis (gam etes) II (gametes) H a p l o i d o r d i p l o i d? C h r o m o s o m e s d u p l i c a t e d o r u n d u p l i c a t e d? C i r c l e o n e g a m e t e f r o m e a c h p a r e n t. T h e s e t w o g a m e t e s u n d e r g o f e r t i l i z a t i o n c r e a t i n g a n e w i n d i - v i d u a l 138 Z y g o t e H a p l o i d o r d i p l o i d? C h r o m o s o m e s d u p l i c a t e d o r u n d u p l i c a t e d?

139 Mendelian Genetics 2. The genotype of a cell is the actual allele combination in that cell. We generally describe genotypes of diploid cells as homozygous dominant (containing 2 dominant alleles), homozygous recessive (containing 2 recessive alleles) or heterozygous (containing 2 different alleles). What allele(s) can be passed into a gamete by Parent One? What allele(s) can be passed into a gamete by Parent Two? 3. In your own words, explain why an understanding of meiosis is necessary before you begin genetics. 4. Complete this Punnett Square depicting the cross between a mother who is heterozygous for gene A and a father who is homozygous recessive for gene A. Note: capital letters usually represent the dominant allele, while lower case letters usually represent the recessive allele. Label where the mother s gametes are listed and where the father s gametes are listed. What do the genotypes listed in the boxes represent? 139 What is the ratio of genotypes you would get for this cross?

140 Mendelian Genetics 5. Remember that a Punnett Square is just a prediction. Suppose a cross between these individuals produces 600 individuals with the genotype Aa and 400 individuals with the genotype aa. Do your results match your prediction? Explain. The primary focus of lab will be to practice using the Punnett square to predict potential offspring genotypes and phenotypes when provided with parental genotypes for a variety of types of inheritance. We will only be practicing with monogenic traits, those for which the phenotype is determined by a single gene. Please keep in mind that most traits are polygenic (phenotype is determined by more than one gene) and complex (an interaction between genotype and environment). Exercise 2: Use of a Punnett Square When listing genotypes, please use the vocabulary homozygous dominant, homozygous recessive, and heterozygous. Carrier describes the genotype of unaffected individuals who have one copy of the disease allele. Autosomal Dominant Dominant traits are those that are expressed in the phenotype if there is at least one copy of the dominant allele. Dominant alleles can phenotypically mask the presence of a recessive allele. In the case of dominant traits, the homozygous dominant and heterozygous genotypes will have the dominant phenotype. Autosomal means that the gene is carried on a non-sex chromosome. Retinoblastoma is an autosomal dominant disease. Retinoblastoma causes eye tumors in about 1 in 20,000 children. R represents the retinoblastoma allele and r represents the normal allele. 1. Answer the following questions about a cross (or mating) between a father who is rr and a mother who is RR. What is the genotype of the father called? What is the genotype and phenotype of the mother? What allele(s) can be passed into a gamete by the father? What allele(s) can be passed into a gamete by the mother? Complete a Punnett square for a cross between these two individuals. 140

141 Mendelian Genetics Provide the probability of each of the possible genotypes and phenotypes. 2. Answer the following questions about a cross (or mating) between a father who is Rr and a mother who is Rr. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 141

142 Mendelian Genetics Autosomal Recessive Recessive traits are those that are expressed only if there are no dominant alleles present. In the case of autosomal recessive traits, only the homozygous recessive genotype will have the recessive phenotype. For recessive traits, individuals who are heterozygous are referred to as carriers. This is because there is not a phenotypic expression of the trait, but the allele does exist and can be passed on to offspring. Cystic fibrosis is an autosomal recessive disease. The cystic fibrosis allele causes an abnormal chloride ion channel. This abnormally-functioning channel results in high levels of production of viscous mucus, which can lead to increased respiratory infections. C represents the normal allele. c represents the cystic fibrosis allele. 1. Answer the following questions about a cross (or mating) between a father who is cc and a mother who is CC. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes.. 2. Answer the following questions about a cross (or mating) between a father who is Cc and a mother who is Cc. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 142

143 Mendelian Genetics Codominance In situations of codominance, there are two or more different dominant alleles. Because both alleles are dominant, both are expressed in the phenotype. This type of dominance is displayed in the ABO blood group. In this case, I A and I B are both dominant, while i o is recessive. The allele I A results in the expression of the type A glycoprotein on blood cells, the allele I B results in expression of the type B glycoprotein on blood cells, and the allele i o results in expression of neither A nor B glycoproteins (type O). There are many different heterozygous possibilities, each of which will have a different phenotype. The heterozygous I A I B will have type A glycoproteins and type B glycoproteins (type AB). The heterozygous I A i o will only express type A glycoproteins (type A). The heterozygous I B i o will only express type B glycoproteins (type B). 1. Answer the following questions about a cross (or mating) between a father who is i o i o and a mother who is I A I B. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes 2. Answer the following questions about a cross (or mating) between a father who is I A i o and a mother who is I B i o. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 143

144 Mendelian Genetics Incomplete Dominance In situation of incomplete dominance, the heterozygous condition has a phenotype that is unique or intermediate between the recessive condition and the dominant condition. This type of inheritance is seen with the sickle-cell allele. Hemoglobin is an important protein in red blood cells. It functions to carry oxygen from the lungs to the tissues of the body. Sickle-cell anemia is a recessive trait that results from a single base substitution. When an individual has two copies of the sickle allele, they will be affected with anemia (low number of functioning red blood cells). S represents the normal allele for hemoglobin. s represents the sickle allele for hemoglobin. SS individuals have a normal phenotype. ss individuals have a sickle-cell anemia phenotype. Ss individuals have a sickle cell trait phenotype. 1. Answer the following questions about a cross (or mating) between a father who is ss and a mother who is SS. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 2. Answer the following questions about a cross (or mating) between a father who is Ss and a mother who is Ss. What is the genotype of the father? What is the genotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 144

145 Mendelian Genetics 3. Answer the following questions about a cross (or mating) between a father who is Ss and a mother who is SS. What is the genotype of the father? What is the genotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 145

146 Mendelian Genetics X-linked Disorders Traits carried on the sex (X or Y) chromosomes have a different inheritance pattern than those on the autosomal chromosomes. Although the X and Y chromosomes line up in meiosis I, they are not homologous and do not carry the same genes. For this reason, there is often only one allele present for a trait rather than two. X-linked disorders are carried on the X chromosome. Females have two copies of the X chromosomes, while males only have a single copy of the X chromosome. The cells of males only contain a single X chromosome; therefore, they cannot be considered homozygous or heterozygous for that allele. Instead, they are considered hemizygous, meaning that they only carry a single allele for that gene. In X-linked recessive disorders, this results in males being more likely affected compared to females. X-linked Dominant For X-linked dominant traits, if the allele is present, it is expressed. Hypophosphatemic rickets is an X-liked dominant disease that causes rickets (weakening of the bone due to abnormal ossification). X R represents the hypophosphatemic rickets allele. X r represents the normal allele. 1. Answer the following questions about a cross (or mating) between a father who is X r Y and a mother who is X R X r. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 146

147 Mendelian Genetics 2. Answer the following questions about a cross (or mating) between a father who is X R Y and a mother who is X R X r. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. X-linked Recessive X-linked recessive traits are more likely to be expressed in males than in females. This is due to the hemizygous genotype. If only a single allele is present and that allele is recessive, it will be expressed. In females, there are two alleles, and a dominant allele can mask the recessive allele. Hemophilia A affects anywhere between 1 in 5,000 and 10,000 males. Hemophilia is caused by a lack of blood clotting factors. This can result in excessive bleeding that does not slow at a normal pace. Without transfusions of clotting factors, fatality often occurs in the early 20s. X H represents the normal allele. X h represents the hemophilia A allele. 1. Answer the following questions about a cross (or mating) between a father who is X h Y and a mother who is X H X H. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 147

148 Mendelian Genetics 2. Answer the following questions about a cross (or mating) between a father who is X H Y and a mother who is X H X h. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 3. Answer the following questions about a cross (or mating) between a father who is X H Y and a mother who is X H X H. What is the genotype and phenotype of the father? What is the genotype and phenotype of the mother? Complete a Punnett square for a cross between these two individuals. Provide the probability of each of the possible genotypes and phenotypes. 148

149 Mendelian Genetics Exercise 3: Probability Probability = number of specific events total number of events When calculating the probability of consecutive events, you must multiply the probability of the individual events. Events that have already occurred do not affect probability. 1. What is the probability that a couple will have a male child? 2. What is the probability that a couple will have 2 male children? 3. What is the probability that a couple will have 4 male children? 4. If a couple already has 3 male children, what is the probability that their next child will also be male? 149

150 Mendelian Genetics Mendelian Genetic Study Guide Questions Answering these questions will be helpful in preparing for your final. 1. What is a gene? 2. What are alleles? 3. Define phenotype and genotype. 4. What is meant by the terms dominant and recessive? 5. In solving genetics problems, how are letters used to designate dominant and recessive. 6. Explain the genotype and phenotype of homozygous dominant, heterozygous, and homozygous recessive individuals. 7. Explain the law of segregation. 8. Explain the law of independent assortment. 9. Explain the inheritance of codominant alleles. 10. How does incomplete dominance affect phenotype? 11. Explain the inheritance of X-linked traits. 12. Why are only females able to be carriers for X-linked recessive traits? Why are males not able to be carriers? 13. When given a genetics problem concerning autosomal dominant, autosomal recessive, codominant, incomplete, or X-linked inheritance, be able to give the genotypes of the parents and correctly use a Punnett square to predict the genotypes and phenotypes of their offspring. 150

151 Guide to Writing a Paper 151 Appendix A: Guide to Writing a Scientific Paper Although writing a scientific paper can seem to be quite a daunting task, writing about your research project or laboratory experiment can give you valuable experience. It helps you learn to think like a scientist and improves your ability to understand scientific papers written by other researchers. Fortunately, there are some common rules and conventions for organizing and presenting information that can make writing a scientific paper about your experiment much easier. The basic structure, rules, and conventions usually apply to scientific writing whether the subject involves biology, physics, or chemistry. Typically, a scientific paper is organized into the following sections: title, abstract, introduction, materials and methods, results, discussion, and literature cited. Title The title of a scientific paper should very specific and informative. This is not the place for a clever, catchy title. Your title should give the reader the important concepts explained in your paper. If appropriate, it should also contain the scientific name of the organisms involved in the study. Often, writers have a working list of titles while writing the paper and decide on the best title once the paper is completed. Below the title put your name as well as the name of the other people in your group who worked on the experiment. Your name goes first (because you are the author), followed by your collaborators. Examples Poor title: Fungus Among Us! (Cute but tells the reader nothing) Better title: Effectiveness of Tea Tree (Malaleuca alternifolia) Oil in Inhibiting the Growth of the Fungus, Candida albicans. Abstract An abstract is a very short summary of both the experiment and the conclusion. Its purpose is to help the reader quickly extract the main points of the paper. That allows readers to assess whether or not the paper is relevant to their research and worth reading in its entirety. Abstracts are usually 250 words long but remember that your paper will be based on an experiment of much smaller scale so you may find that 3-6 sentences summarize your work very well. Many writers find that it s easier to write the abstract after they have completed the rest of the paper. Introduction This section sets the stage for the rest of the paper. It provides background information to help the reader understand your topic, describes prior work related to your experiment, and tells the reader the reason why the paper was written. It is best to begin with a more general discussion and then narrow down to your specific experiment. Near the end of the introduction, explain the rationale (reason) for your work by relating it to the background information you gave earlier in the introduction. You want to link your experiment to what is

152 Guide to Writing a Paper already known about the topic (a very focused literature review) and give your reader a clear idea of your objectives (what question did you try to answer, what were you trying to learn?). Conclude your introduction with a statement of purpose and the hypothesis that your experiment tested (see examples below). Briefly mention, in two or three sentences, how you conducted your experiment. Remember to cite your references in the body of the introduction for all information that is not due to your own personal discovery. Direct quotes are not appropriate in a science research paper. There will be more information about how to cite references later in this guide. Materials and Methods In this section, you will describe exactly how you conducted your experiment. It must be written in past tense and in paragraph form. Past tense means that you write was heated instead of is heated. Paragraph form means that you don t make a list of equipment. Instead, you write a paragraph explaining exactly how you did your experiment, including the equipment, solutions, etc., as appropriate. Scientific papers are generally written in passive voice, although in the course of your research, you may come across a few papers written in active first person. Passive voice means that you write the solution was heated instead of I heated the solution. Ask your professor what form he or she prefers and then be consistent. Either way, you want to be as clear and precise as possible. Write your materials and methods section so that anyone could repeat the experiment properly based on your report. You do need to include the details of the equipment used, concentration of solutions, and so on. You do not need to tell your reader to label or to wear safety goggles because you assume you are writing for a professional audience. Do not copy the instructions in your lab manual word for word. You do, however, need to cite your lab manual because it is your source of information as to how to do this kind of an experiment. Do not mention the results in this part; that s for the next section. Results Here, you present your data. Be sure to include data that does not seem to fit. Don t automatically conclude that you made a mistake. Just put it out there. You will present your data in two forms, in table or figures (such as graphs) and in paragraph form. The tables or figures are often placed at the end of the paper, after the conclusion but before the Literature Cited section. Tables and figures must be numbered, have a title, and be properly labeled. Make sure that you have the correct units, axes labeled on the graph, etc. Whether you choose a table or a figure depends on which best depicts your results. For the paragraph, write as though you are presenting the results to someone who cannot see your tables or graphs. However, if you have figures and tables that accompany your sentences, make sure you let your reader know when to look at them. For example, The bean plants in the control plot grew an average of 50 ±2.5 cm (Figure 1). For the purposes of this paper, just present the data (the numbers) but do not interpret or explain them. Your ideas about what the data mean will be presented in the discussion. Just tell what happened without comments in the results section. Do not include any raw data. 152

153 Guide to Writing a Paper Discussion In the discussion, you wrap everything up. It is helpful to first note your major findings as presented in the results section. Then, explain your results and discuss their implications. Address whether or not your results support your hypothesis. Refer to your tables or graphs as support for your explanations. Compare your results with those of other researchers by discussing your results in the context of further background information and the experiments of other researchers (remember to cite your sources). Also, talk about any inconsistencies, unexpected results, or problems that you encountered, and suggest some explanations and ways you could have improved your experiment. This is also the place to indicate the direction of experiments which could further our understanding of your topic. Conclusion The conclusion is a brief two or three sentence summary of the main points of your discussion. Literature Cited In this part of your paper, list the actual sources you made reference to in your writing. A citation appears in the body of the text of paper wherever you use information from a particular source, and then that source is included on the Literature Cited page. Your list must be alphabetical by author s last name. There are different styles of literature citation. You must use the Council of Science Editors (CSE) name-year style of citation. This format was formerly known as CBE (Council of Biology Editors) format. This guide includes a sample paper with intext citations in this style as well as a Literature Cited section. You can also find a great guide online at Click on the name-year system. Under the name-year system, there is help with citing references within your text as well as help with making a list of references for the Literature Cited section. You can find help about how to cite internet sources using the CSE name-year system by going to our library s home page, clicking Research at the top of the page, then clicking cite your sources under How To. Choose Other Formats, then click citing your sources. On the page that pops up, CSE will be one of the choices across the top of the screen. Scroll down to the name-year system. There you will find information about how to cite internet sources and other sources as well. 153

154 Guide to Writing a Paper General Requirements for the Paper: Information must be organized and presented as detailed in the Guide for Writing a Scientific Paper (Appendix A). All references must be cited within the body of the paper for all information that is not your original idea and also included in a Literature Cited section, presented in the CSE name-year style. A minimum of three sources, one of which must be from a peer-reviewed scientific journal. Note that this is the minimum requirement; many well-researched papers will have more sources. Also, please note that Wikipedia is not an acceptable source. Times New Roman 12 or Calibri 11 font, double-spaced. Margins should be 1 inch. Paragraphs need to be indented or separated from the preceding paragraph by a space so that it is clear where the new paragraph begins. Make sure that major sections of the paper stand out (bolded, underlined, or italicized). One or two-sides is OK, but two-sides saves paper. No cover jacket (plastic/cardboard report covers). Your instructor may have directions/requirements that vary from those above. Be sure to follow all directions as given by your instructor. The following pages are an example of a a real student paper. It is excellent. However, it is not perfect. If any element of style in the example paper conflicts with the written or verbal instructions as given in your lab, the instructions given in lab are what you should follow. 154

155 Guide to Writing a Paper Sample Scientific Paper Vigilance and the Effect of Group Size on the Ring-Necked Duck (Aythya collaris) and the Northern Pintail (Anas acute) Ashley Reyes (used with permission) Abstract When animals perform tasks where vigilance levels have decreased, they are more susceptible to predators due to a hindered ability to see and escape from the attacking enemy. They must therefore exhibit trade-off behaviors, in which they risk predation for mental and physical nourishment. The purpose of this experiment was to test the effects of group size on vigilance, during fifteen minute time intervals, in the Ring-Necked Duck (Aythya collaris) and the Northern Pintail (Anas acute). The Ring-Necked Duck (Aythya collaris) was sleeping and isolated, while the Northern Pintail (Anas acute) was sleeping and in a group. Moreover, previous experiments have suggested that there is an inverse relationship in vigilance and group size, where vigilance levels decreased with increased group size. It is hypothesized that as an individual increases its distance from the group, vigilance will increase (as will be demonstrated by an increase in the number of eye peeking during sleeping). After the 15 minute time interval, the Ring-Necked Duck (Aythya collaris) peeked one hundred and sixtythree times and the Northern Pintail (Anas acute) peeked sixty-five times. There was a significant difference between the number of times the animal peeked, compared to an individual in solitude and an individual in a group (χ 2 = 42.13, df = 1, P 0.05). Therefore, there is evidence to indicate that the individuals will display a higher level of vigilance, or an increase in the amount of peeking, when in isolation compared to being in a member of a group. 155

156 Guide to Writing a Paper Introduction Vigilance levels and sleeping are often mutually exclusive events when an animal needs to detect predators and conserve energy. Yet, the setting in which these activities take place may influences how much time one task must be compromised for the other. Moreover, when animals do perform tasks where vigilance levels have decreased, they are more susceptible to predators due to a hindered ability to see and escape from the attacking enemy. In order to ensure survival, there must be trade-offs exhibited in animal behavior that weigh the risks of predation against the benefits of rest (Brodin, 2001). These trade-offs often occur during times dedicated to sleeping and times watching for predators, which are instances of high and low risk, respectively. For the most part, the animal must display its greatest level of anti-predator behavior during high-risk situations, which are frequent and brief, and lower its vigilance during low-risk situations (Lima and Bednekoff, 1999). This ensures that the animal can partake in its current activity while ensuring its safety at the same time. While sleeping is vital to all animals, birds perform certain behaviors that help reduce its predator risk. At the individual level, one of a bird s main defenses includes its use of visual scanning. This is important because birds lack other defense mechanisms, such as claws and camouflage, to deter or hide from attack (Franklin and Lima, 2001). To solve the dilemma of being attacked while sleeping, birds alternate between periods of vigilance, where their eyes open or peek, and sleeping, where their eyes are closed (Gauthier-Clerc, et al. 2000). 156

157 Guide to Writing a Paper At the group level, birds use increasing group size as a deterrent to predator attacks. In experiments by Lima, et al. (1999), who observed free-living Dark-eyed Juncos (Junco hyemalis), there is data suggesting that there are great benefits of high sociability since the vigilance of group members is combined. Otherwise known as the many eyes effect, there is an improvement in the detection of predators, since there are more eyes with which to detect attack. Another preventive strategy of being a member of a large group is risk dilution. Here this situation, the likelihood of an individual being harmed decreases because there are a greater number of other individuals that the predator can attack (Beauchamp, 2001). Some members of the group are also known to display a geometric selfish herd effect where animals in the center of the group decrease their predation risk by surrounding themselves with others (Uetz, et al. 2002). Other factors that affect the group size affect are the amount and extent of visual obstructions, the distance to the closest safe refuge, and the abundance and type of predator (Lima and Bednekoff, 1999). Yet, there are also consequences to increasing group size. This can result in the competition hypothesis, where even though the larger group has an increased ability to find food (McNamara and Houston, 1992), the larger group increases competition for this limited resource since there is now more time to eat a greater amount of food (Lima, et al. 1999). The purpose of this experiment was to test how group size affected vigilance in the Ring-Necked Duck (Aythya collaris) and the Northern Pintail (Anas acute). It is hypothesized that as an individual increases its distance from the group, vigilance will increase as will be demonstrated by an increase in the number of eye peeking during sleeping. 157

158 Guide to Writing a Paper Materials and Methods The Ring-Necked Duck (Aythya collaris) and the Northern Pintail (Anas acute) were each observed at the Oregon Zoo in Portland, Oregon, on February 22, The temperature was 8 C and the sky was overcast with occasional gusts of wind. During the observance time period, a stopwatch was started and then stopped before and after fifteen minutes. A duck was considered to be peeking if its eyes were closed and its eyelids opened all of the way, revealing the entire eye. The number of peeks only counted if they occurred within the given time period. In order for a duck to be considered sleeping, its beak must have been ticked into its feathers, as though it were preening with its beak tucked below its right or left wing, and it feet stayed immobile before and during the given time period. The individual was still considered to be a testable subject even if it stretched its wing or brought its head up out of sleeping position for less than fifteen seconds. Otherwise, the individual was not considered to be sleeping, and thus wasn t peeking. The individual was considered isolated if it did not react to or react with another individual, stayed at least two foot away from other individuals, and was the only individual sleeping during the given time interval. The individual was considered to be apart of a group if reacted with or reacted to nearby individuals who were also considered to be sleeping during the given time interval. The first animal to be observed was the Ring-Necked Duck (Aythya collaris), between 10:04 am and 10:19 am. The isolated individual sat on a patch of dry dirt, between two small bodies of water. The pool to individual s right was larger than the pool on the individual s left. The duck was secluded, except for the five other ducks were swimming in the larger, or higher elevated, body of water. One foot behind the duck was a waterfall, indicating a change of 158

159 Guide to Writing a Paper elevation between the bodies of water. There was approximately a one foot gap. Two feet in front of the duck was a cement wall. The second animal to be observed was the Northern Pintail (Anas acute), between 11:05 and 11:20 am. The individual sat on secluded piece of land, surrounded by water on three of the four sides with three other ducks, all of whom were also sleeping. The individual observed was the third duck furthest from the left, where it was in the inside of group. A Chi-square Goodness-of-fit test was performed in order to determine the data s significance. Results After separate independent fifteen minute time intervals, the Ring-Necked Duck (Aythya collaris) opened its eyes to one hundred and sixty-three times while the Northern Pintail (Anas acute) peeked only sixty-five times. This data is physically represented in Figure 1. There was a significant difference between the number of times the duck peeked, compared to an individual in solitude and an individual in a group (χ 2 = 42.13, df = 1, P 0.05). Discussion The Ring-Necked Duck (Aythya collaris) displayed a greater level of vigilance than the Northern Pintail (Anas acute). There is evidence suggesting that individuals display a higher level of vigilance, or an increase in the amount of peeking, when in isolation compared to an individual group member. The data is consistent with the findings of Lima, et al. (1999), who also experimentally determined that vigilance levels decreased with increasing group size with free-living Dark-Eyed Juncos (Junco hyemalis). Since the Ring-Necked Duck (Aythya collaris) lacked the many eye benefits, it was forced to dedicate less time for sleeping and more time to peeking and scanning for predators. The Northern Pintail (Anas acute) had the security, 159

160 Guide to Writing a Paper however, of being able to sleep more and peek less, since there were three other members of the group that also displayed vigilant peeking. There are, however, some inconsistencies in the data that weaken the validity of the results. Due to discrepancies in the behaviors and habitat of the Ring-Necked Duck (Aythya collaris) and the Northern Pintail (Anas acute), the results after the fifteen minutes of observation might not be compatible between the species. It was acknowledged the data is not very significant due to the short observance period and the fact that two different species were analyzed. More studies should be done to further elucidate the relationship between group size and vigilance in sleeping duck species Number of Peeks Ring-Necked Duck Aythya collaris (one member) Northern Pintail Anas acute (4 members) 40 0 Group Size Figure 1. Group Size and vigilance were quantitatively measured and compared in the Ring-Necked Duck (Aythya collaris) and Northern Pintail (Anas acute) using the number of eye peeks during sleeping. In the fifteen minute interval, the Ring-Necked Duck (Aythya collaris) peeked 163 times and Northern Pintail (Anas acute) peeked 65 times. 160

161 Guide to Writing a Paper Literature Cited Brodin A Mass-dependent predation and metabolic expenditure in wintering birds: is there a trade-off between different forms of predation? Animal Behaviour. 26: Beauchamp G Should vigilance always decrease with group size? Behavioral Ecology and Sociobiology. 51: Gauthier-Clerc M, Tamisier A, Cezilly Sleep-vigilance trade-off in gadwall during the winter period. The Condor. 102: Lima SL, Bednekoff PA Temporal variation in danger drives antipredator behavior: the predation risk allocation hypothesis. The American Naturalist. 153: Lima SL, Franklin WE Laterality in avian vigilance: do sparrows have a favourite eye? Animal Behaviour. 62: Lima SL, Zollner PA, Bednekoff PA Predation, scramble competition, and the vigilance group size effect in dark-eyed juncos (Junco hyemalis). Behav. Eco.Sociobio. 46: McNamara JM, Houston AI Evolutionary stable levels of vigilance as a function of group size. Animal Behaviour. 43: Uetz GW, Boyle J, Heiber CS, Wilcox RS Antipredator benefits of group living in colonial web-building spiders: the early warning effect. Animal Behaviour. 63:

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163 Guide to Scientific Figures Appendix B: How to Make a Scientific Figure After completing your experiments, collecting, and analyzing your data, your next task is to prepare the data for presentation. Figures and tables should simplify the information you want to report, and illustrate the key findings. When choosing whether to use a table or a figure (graph) to represent your data, think first about the type of data that you have collected and the type of information that you want to convey. Figures, such as graphs, are the most widely used form of illustration in science, but some types of data are more appropriately shown in table form. You usually will not present your raw data, but rather a summary of the analyzed data. Tables are useful to present lists of numbers or text in columns, each column having a title or label. Figures are more useful for showing a trend or pattern of relationship between sets of values. To plan the layout of your table or figure, you must first identify your variables. Variables can be classified in different ways. The independent variable is the quantity or category that is subject to manipulation or choice by the investigator. You are in control of these and have chosen these. Examples are time, distance, temperature, ph, or concentration. The independent variable data is plotted on the X-axis (the bottom, horizontal axis). The dependent variable is a measured property that varies as the independent variable is changed. This refers to measurements that you are taking, such as temperature measurements under different conditions, counts per behavioral category, blood pressures or responses to each treatment category. A data series or data set is a group of measurements that correspond to the values or categories of an independent variable. It is usually the relationship between the independent and dependent variable that is of interest. This relationship is what your figure (or table) should illustrate in the most concise and organized manner. Your data can also be continuous or discontinuous. Continuous data is data where the underlying distribution of data is continuous and the discrete points of data are the results of an arbitrary scale imposed by the investigator, i.e. human height or weight. We can measure height in centimeters and then plot the height of each subject; but in the populations at large, between each subject, there will always be someone whose height is just a bit higher or lower than our data point, so a line graph is an appropriate tool to represent the continuous nature of the data. Discontinuous data is data where no intermediate values are possible. The independent variable points are distinct groups (males vs. females) or categories (different species) or discrete behaviors, such as right handed vs. left handed behaviors. Since there are no intermediates between these categories, it would be inappropriate to make a line graph with the data (a line indicates a continuous range of data). A more appropriate way to represent this type of data set is a bar graph, table or pie chart. 163

164 Guide to Scientific Figures After you have chosen the appropriate format for presenting your data and made the tables and/or figures, you will assign the figures and/or tables numbers. Label the tables and figures independently of each other (Table 1, Table 2, Figure 1, Figure 2) in the sequence in which you refer to them in the text. It is helpful to the reader to place the figure or table near the text reference. Each figure or table must be mentioned in the text. Both tables and figures require a clear and complete legend or caption. The legend should convey as much information as possible about the table or figure. It should give enough information that the reader can understand the figure without reading the text. Elements of a Typical Table: Table 1. Root growth of selected plants grown. 164 Summer 2005 in Portland, Oregon* Table legend Time Ethronium Saxifraga Arnica Column titles (days) oregonium tolmiei mollis Table body (data) * Length, in cm footnotes * Plants were grown in a hydroponic solution Lines demarcating the different parts containing 3% nitrate, 2% soluble potash, 3.2% calcium and.04% chelated iron from for 12 days, 10 plants/group. Guidelines for a Table: of the table A period is placed after the table #. The legend is placed above the table. Tables should report summary level data rather than raw data. Units should be specified (either in the column headings or a footnote). Footnotes are used to clarify points in the table or data. Lines of demarcation are used to separate the legend, column headings, data and footnotes. Tables are useful to present lists of numbers or text in columns with each column having a title or label.

165 % Cumulative germination BI 112 Guide to Scientific Figures Figures (xy plots, graphs) are visual way to show a trend or a pattern of relationship between sets of values. Elements of Typical Graphs: Y axis Presoaked Controls Data set and symbols Key to symbols Y axis label 0 Origin Time (days) Figure legend Figure 1. Percent cumulative germination of Quercus rubra seeds after pretreatment with a 2 day soak in 5% NaCl. Control seeds soaked for 2 days in water. n = 1 trial per treatment group (100 seeds/trial). Guidelines for a Figure (XY Plots, Bar Graphs): X axis label X axis A period is placed after the figure # and also the legend itself. The term Figure can be abbreviated to Fig. in the text, but should never be abbreviated in the legend or caption. The legend goes below the figure. Each legend should convey as much information as possible about the figure as possible: the subjects of the experiment, the treatment applied or the relationship displayed, location (if a field experiment), and sample sizes and statistical tests if they are not displayed elsewhere. A legend box should explain the symbols associated with each data set. Choose symbols that are a simple form and can be easily differentiated by the reader. The axes are the horizontal and vertical lines that define the plot area. The vertical (y) axis always represents the dependent variable (measured data). The horizontal (x) axis always represents the independent variable (these are the ones you chose). Each axis should have a label indicating what is being plotted and the units. 165

166 % Mortality BI 112 Guide to Scientific Figures Choose a scale for your figure that is appropriate for your data set. You do not want to have your data points off the scale, but you also do not want the plot area to have a large blank space with your data scrunched in a corner. Look at your highest and lowest data point to choose a scale that distributes your data appropriately. Everything should be clearly labeled and easily discernable to the reader. The reader should not have to squint to see any of the information. Example of a Bar Graph: Bar graphs are useful when you want to compare the value of a single variable (usually a summary value, such as the mean values) among several distinct categories or groups, as in the example below. Y axis label Y axis Male Sex Female X axis label ph 5 ph 7 ph 9 x axis Figure 2. Effects of water ph on % mortality of male and female Paramecium caudatum incubated in 10 ml of spring water adjusted to either ph 7, 8, 9 or 10 for 50 min. at 26 C. n= 3 trials, 25 P. caudatum per treatment group. Figure legend 166

167 Guide to Scientific Figures Scientific Figure Study Guide Questions Be able to answer for a quiz. 1. Should the independent variable go on x or the y axis? 2. Should the dependent variable go on the x or the y axis? 3. What are continuous data? 4. What are discontinuous data? 5. How detailed should a figure or table legend be? 6. Where should the figure legend go on a table? On a figure? 167

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169 Appendix C: Microworlds Project Instructions The purpose of this project is to allow you to become very familiar with the use of the microscope before you get into classes (such as microbiology) where microscope use will be assumed. To this end, you will be expected to complete roughly two Microworlds entries per week. 1. You are required to complete a minimum number of Microworlds entries. There are two entries per page. 2. Check with your instructor regarding the due dates and requirements for the Microworlds project. 3. If anything in the following instructions or examples conflicts with the written or verbal instructions given in lab, the instructions given in lab are what you should follow. 4. Please carefully review the requirements for the Microworlds project below: At least FOUR entries must be done using the oil immersion lens. At least TWO entries must be done using slides that you made yourself (not preprepared slides; for example: cheek cells, Elodea leaf sample, yeast, etc). In the circle at the top of the page, make a careful and accurate drawing of what you actually see through the microscope. You may color the drawing, but coloring is not required. Name of specimen = what you are looking at. If it is a prepared slide, write what it says on the slide. If you made the slide, write a detailed description of what you are looking at (e.g. human cheek cell ). Kingdom = the Kingdom the specimen belongs to. If you are unsure, look it up on the internet. Total magnification = magnification of objective x magnification of eyepiece Field size = how large the field of view is. You measured this in lab on the first day; use those measurements! Estimation of object size = do your best to estimate the size of the entire specimen or a part of it. If the part you are measuring is not immediately obvious, label it on your drawing. Key to labelled structures = label a minimum of two things on your drawing. If you don t know what any structures are, look up the name of your specimen on the internet. If you still don t know, do your best to try to identify something. Draw a line from the structure out to your label (which may be abbreviated). 169

170 Microworlds Project Example CM cm N sp Name of specimen: Human Cheek Cell Kingdom: Animalia Total magnification: 400X Field size: 500μm Estimation of object size (be sure to indicate the object): 1 cell = 200μm in length Key to labelled structures: (e.g. cw = cell wall, n = nucleus, etc.) Add additional items as necessary 1. cm = cell membrane 2. n = nucleus Name of specimen: Spirilli (spiral shaped bacteria) Kingdom: Eubacteria Total magnification: 1000X (oil) Field size: 200μm Estimation of object size (be sure to indicate the object): length of 1 bacteria = 50μm Key to labelled structures: (e.g. cw = cell wall, n = nucleus, etc.) Add additional items as necessary 1. cm = cell membrane 2. sp = spiral-shaped bacteria (no other structures can be seen)