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Physiology 3 Lab Manual, Spring 2012 1 Physiology Laboratory Manual Spring 2012 Dr. Christina G. von der Ohe Santa Monica College Physiology 3 Lab Manual, Spring 2012 2 Table of Contents Course Material Page Syllabus ……………………………………………………………………………………………………… 3 Calendar ……………………………………………………………………………………………………. 6 Instructions for lab notebooks …………………………………………………………………… 7 Instructions for formal lab reports ……………………………………………………………. 8 Example lab report ……………………………………………………………………………………. 9 Group presentation instructions ………………………………………………………………… 12 Lab 1: Skills lab …………………………………………………………………………………………. 13 Lab 2: Homeostasis ……………………………………………………………………………………. 22 Lab 3: Enzymes …………………………………………………………………………………………. 24 Lab 4: Osmosis ………………………………………………………………………………………….. 28 Lab 5: Neurobiology ………………………………………………………………………………….. 31 Lab 6: Sensory Physiology …………………………………………………………………………. 34 Lab 7: Article Review…………………………………………………………………………………. 39 Lab 8: Digestive System …………………………………………………………………………… 40 Lab 9: Blood ………………………………………………………………………………………………. 43 Lab 10: Cardiovascular Physiology ……………………………………………………………. 45 Lab 11: Urinary System …………………………………………………………………………….. 49 Lab 12: Immune System .………………………………………………………………………….. 51 Physiology 3 Lab Manual, Spring 2012 3 Physiology 3: Human Physiology Instructor: Christina G. von der Ohe, PhD, Professor, Dept. of Life Sciences Office: SC-261 Phone: (310) 434-4662 Email: email@example.com OH: MW 7:30-9:00am, 2:30-3:00pm Meeting Lecture MW 9:30-10:50am SC-151 Times: Lab section 2873: M 11:15-2:20pm SC-201 Lab section 2874: W 11:15-2:20pm SC-201 Student 1. Given a problem or set of conditions, write a hypothesis, Learning provide an experimental design, and identify dependent and Objectives: independent variables, and control and experimental groups. 2. Identify the physiological mechanisms that each body system employs to maintain homeostasis. 3. Students should demonstrate confidence in their understanding of biological concepts and the scientific method to evaluate and critique current media or a scientific report. Textbook: Human Physiology, S. Fox, 10th, 11th, or 12th ed. Physiology Laboratory Manual, C. von der Ohe Required Quad ruled carbonless lab notebook: 8.5 x 11” left spiral bound Materials: 3 scantrons #882 or #25110 and a #2 pencil Ruler, Colored pens or pencils, Calculator Resources: Learning Resource Center and Computer Lab Textbook resources at aris.mhhe.com Recordings: http://smc.kspill.com/pages/phys.html Homepage: http://homepage.smc.edu/vonderohe_christina/ Attendance: Students who are absent for two consecutive meetings or to the first exam without informing the professor with a valid excuse will be dropped from the roster. Drop Dates: Drop dates are listed in your catalog. You are responsible for your enrollment status and the dates and deadlines on the SMC admissions website and schedule of classes. Make-ups: There will be no make-ups for labs or presentations. Only one lecture exam can be made up under extreme circumstances and with instructor consent BEFORE the start of the exam. The make-up exam will be given on the same day as the final. The final exam cannot be made up. Physiology 3 Lab Manual, Spring 2012 4 Lecture: We will start the lecture with an extra credit opening question. The lecture will be based on PowerPoint slides that will be posted to ecompanion before lecture. You are welcome to print the slides before class and take notes on them. Opening At the start of every session you will answer a brief extra credit Question: question based on the previous lecture or current lab. They must be your own work. Lab: In lab, you will design, execute, and record experiments in groups. You are required to read the lab manual before coming to lab. You will be writing each lab into a notebook that must remain in the classroom for the entire semester. The lab reports must be individual and unique work. Bring your textbook and lab manual to each session. There are no make- ups for labs, but I will drop your lowest score. Class I strive to make the classroom a safe and encouraging learning Environment: environment for everyone. There will be a lot of class discussion and group work. Please be respectful of each other. I encourage you to freely ask questions so that everyone can benefit from the discussion. This class is for you. Please turn off all beepers and cell phones during class. Food, drink (including water), and gum are not permitted in any of the science rooms. TBA Hour: You are required to spend one hour per week studying in the Learning Resource Center (LRC): Science Complex 240 & 245. Presentation: There will be group presentations on a recent scientific discovery. This assignment will be discussed in more detail later in the course. Grading: You will be evaluated based on performance on exams, lab reports, a group presentation, and attendance and participation. Points will be totaled and expressed as a percent. Grades are non-negotiable and must be earned. 3 lecture exams 300 90-100% = A 1 final exam 200 80-89% = B 12 lab reports (1 dropped) 275 70-79% = C Group presentation 75 60-69% = D Participation 35 Below 60% = F TOTAL POINTS 885 At the end, your lecture exam scores will be averaged and your lowest score will be replaced by the average. Missed exams do not qualify for this. Physiology 3 Lab Manual, Spring 2012 5 Exams: Lecture exams consist of multiple choice, true/false, and short answer questions. The correct scantron is required. Lecture exams are not cumulative. The final exam consists of short answer questions and is cumulative. All books, notes, and electronic devices must be put away before each exam. Use the restroom before the exam; you may not leave the room until you are finished with your exam. Exam We will review the exam in lab. The exams are my property, Viewing: and may not leave the classroom in any form. I will return your exam to you and post the answers at the side of the classroom. You will have 15 minutes to review your exam and ask questions. You may not take notes, photograph, or leave the room with the exam. Any of these offenses will result in the filing of an Academic Dishonesty report and a loss of points on the exam. If you need more time to review the exam, you are welcome to view it in my office. To succeed in Physiology 3 is a very rigorous class that requires considerable this class: discipline, time, and dedication. Tips for success: 1. Arrive to class on time. 2. Be well rested and alert for class. 3. Be prepared for exams. 4. Keep track of your grades. 5. Practice effective study habits: - study 30 min to 1 hour every day - recite the material and draw structures from memory - engage in class and visit my office hours I value: 1. Interest in the material 2. Hard work 3. Respect for everyone in the classroom 4. Integrity in your work 5. Responsibility for your grade 6. Punctuality Academic You must do your own work on all opening questions, exams, Dishonesty: and lab reports. A first offense of academic dishonesty will result in a zero on that material and the filing of an Academic Dishonesty Report. A second offense in the college or an egregious offense will result in disciplinary action. Please refer to the SMC policy on academic dishonesty. Final Word: If you have any questions about course material, computer, internet, campus resources, future plans, or anything else, please don’t hesitate to ask. I am here to help you. Physiology 3 Lab Manual, Spring 2012 6 DATE TOPIC FOX CHAPTER LAB Feb 13 Introduction & Homeostasis 1 Skills Lab Feb 15 Chemistry 2 Skills Lab Feb 20 HOLIDAY Feb 22 Cell Biology 3 No lab Feb 27 Enzymes 4 Enzymes Feb 29 Cellular Respiration 5 Enzymes Mar 5 Transport Mechanisms 6 Osmosis Mar 7 Muscle, Review 12 Osmosis Mar 12 Lecture Exam 1 Group Projects Mar 14 Intro to Neurobiology 7 Group Projects Mar 19 Autonomic Nervous System 9 Nervous System Mar 21 Central Nervous System 8 Nervous System Mar 26 Sensory Physiology 10 Senses Mar 28 Endocrinology 11 Senses Apr 2 Reproductive System 1 20 Article Review Apr 4 Reproductive System 2 20 Article Review Apr 9 SPRING BREAK Apr 11 SPRING BREAK Apr 16 Digestive System 18 Digestive Apr 18 Growth + Metabolism, Review 19 Digestive Apr 23 Lecture Exam 2 Blood Apr 25 Cardiovascular System 1 13 Blood Apr 30 Cardiovascular System 2 13 Cardiovascular May 2 Cardiovascular System 3 14 Cardiovascular May 7 Respiratory System 1 16 Presentations May 9 Respiratory System 2 16 Presentations May 14 Urinary System 1 17 Urinary System May 16 Urinary System 2 17 Urinary System May 21 Immune System 15 Immune May 23 Review Immune May 28 HOLIDAY May 30 Lecture Exam 3 Discussion Jun 4 Review No lab Jun 11 Final Exam 8-11am Physiology 3 Lab Manual, Spring 2012 7 Lab Notebook In this course, you will be recording your experiences into a lab notebook that will remain in the lab classroom for the entire semester. The objective of this exercise is to record and communicate your physiology learning experience. Your reports must be written during the lab period and must be complete by the end of each lab session. Lab Must be quad ruled, carbonless, 8.5 x 11”, spiral bound on the notebook: left side, available at bookstore. Write your name on the front of your notebook. All labs must be written in pen. Instructions: Every lab has unique instructions for the lab report, which can be found under the “Lab Notebook” heading. Make sure you follow these instructions in order. The labs will be performed in groups, but your reports must be individual and unique work. Any shared or copied entries will receive a zero grade. You are welcome to tape or staple anything into your lab notebook. Reviewing At the end of each lab period, I will post the answers in the your lab: front of the room. This may only be viewed after your lab report is complete and you have handed in your notebook. The answers are my property; do not copy or photograph them, and do not share them with other students. Copying the answers, adjusting your lab report, or sharing answers with other students will be considered a violation of the Student Honor Code, and will be handled accordingly. Grading: Your lab notebooks will be graded 3-4 times during the semester. Any work that is not affixed to your lab notebook will not be graded. Physiology 3 Lab Manual, Spring 2012 8 Formal Lab Reports In this course, you will be instructed to write 2 formal lab reports. Follow this format for each formal lab report: 1. Date: 2. Title: Make sure that the title is descriptive. 3. Introduction: A 1-3 paragraph explanation of the physiology being explored in the lab. Use complete sentences. 4. Purpose: State the purpose(s) of the lab. 5. Materials: List all of the materials you use. Avoid unnecessary details. 6. Procedure: Briefly list the steps that were actually taken. List statements in past tense. Do not use personal pronouns. 7. Data: This section contains the raw data results of the experiment. It should also include a table or graph. If using a graph, it must be labeled, and must be made with a ruler. 8. Discussion: Using complete sentences and paragraph format, explain the results. What do your observations tell you? How does this fit into the context of the physiological concept being explored? What is probable mechanism behind your findings? How robust are the results, given their statistical and biological significance? What are potential sources of error? 9. Questions: Answer any questions posed in the lab manual. Physiology 3 Lab Manual, Spring 2012 9 Sample Formal Lab Report 1. Date: August 12, 2007 Lab partners: Harry Potter, Hermine Granger, Ron Weasley 2. Title: Enzymes and temperature affect chemical reactions 3. Introduction: Chemical reactions are important in biology because they allow molecules to be formed and destroyed. There are four ways in which a chemical reaction can be sped up: heat, increasing the concentration of reactants, decreasing the concentration of products, and the presence of enzymes. Enzymes are proteins that catalyze reactions. They do so by lowering the activation energy for a reaction. Enzymes are important in regulating chemical pathways, synthesizing materials needed by cells, releasing energy, and transferring information. They play a role in every system in the body. Enzyme activity can be affected by temperature, pH, and the amount of reactant or product. This lab focuses on the activity of the enzyme veritase in making the truth potion “veritaserum.” A successful veritaserum is gold in color. 4. Purpose: 1. To learn the role of the enzyme veritase in making the truth potion “veritaserum.” 2. To understand how the activity of veritase is affected by the temperature of the cauldron. Experiment 1 5. Materials: 2 cauldrons, stirring spoon, knife, wormwood, root of asphodel, eye of newt, hair of wooly spider, 2 drops of veritase, goldmometer 6. Procedure: Finely cut wormwood and root of asphodel Place the following ingredients in each caudron: 10g wormwood, 5g root of asphodel, 2 eyes of newt, and 1 hair of wooly spider In one cauldron, add 1 drop of veritase Stir each mixture counterclockwise for 1 minute Record the color of each potion each minute for 5 minutes using the goldmometer (1=gray, 2=brown, 3=gold) 7. Data: 1 minute 2 minutes 3 minutes 4 minutes 5 minutes - veritase 1 1 1 1 2 + veritase 1 1 2 3 3 Physiology 3 Lab Manual, Spring 2012 10 Activity of veritase 4 Goldmometer reading 3 (units) - veritase 2 + veritase 1 0 1 2 3 4 5 Time (minutes) Experiment 2 6. Materials: 2 cauldrons, stirring spoon, knife, thermometer, wormwood, root of asphodel, eye of newt, hair of wooly spider, 2 drops of veritase, goldmometer 7. Procedure: Finely cut wormwood and root of asphodel Place the following ingredients in each caudron: 10g wormwood, 5g root of asphodel, 2 eyes of newt, and 1 hair of wooly spider, 1 drop of veritase Heat one cauldron to 60ºC Stir each mixture counterclockwise for 1 minute Record the color of each potion each minute for 5 minutes using the goldmometer (1=gray, 2=brown, 3=gold) 8. Data: 1 minute 2 minutes 3 minutes 4 minutes 5 minutes Room temp 1 1 2 3 3 60 ºC 1 2 3 3 3 Physiology 3 Lab Manual, Spring 2012 11 Effect of heat on veritase 4 Goldmometer reading 3 room (units) temperature 2 60 deg C 1 0 1 2 3 4 5 Time (minutes) 9. Discussion: Both the enzyme veritase and heating sped up the chemical reactions in the truth potion “veritaserum.” As you can see from the chart and graph in Experiment 1, the addition of veritase increased the speed of the reaction by 2 minutes. Experiment 2 revealed that heating the potion further speeds up the chemical reaction by 1 minute. Although statistical significance was not determined, the 40% increase in rate with veritase is a substantial magnitude. Thus, these experiments have demonstrated that the enzyme veritase can speed up this chemical reaction, and that heat increases its rate still farther. It is possible that the rate of reaction in the heated cauldron in experiment 2 was even greater than we recorded because a) we only had one eye of newt and b) we were not able to heat the cauldron above 50ºC. With only one eye of newt, we decreased the amount of reactants, which can slow down a chemical reaction. With a cooler cauldron, we also may have slowed down the chemical reaction. 10. Questions: 1. How much sooner did the veritaserum begin to turn gold with veritase, versus without? Veritaserum began to turn gold 2 minutes earlier when veritase was added. 2. Did veritase speed up the chemical reactions taking place in the brewing potion? Yes, veritase sped up the chemical reactions that took place in the brewing potion. 3. How much sooner did the veritaserum begin to turn gold when heated? Veritaserum began to turn gold 1 minute earlier when veritase was added. 4. Did heating the potion speed up the enzymatic reaction of veritase? Yes, heating the potion sped up the enzymatic reaction of veritase. Physiology 3 Lab Manual, Spring 2012 12 Group Presentation 8% of your grade is based on a group project. You will work in groups of 2-3 to explore a new scientific finding and present the finding and its underlying physiology to the class. All groups will present their work during lab at the end of the semester. Your group will be assigned a specific system on which your presentation must be focused. Find an original research article that has been published in the last year. Good sources for finding articles are pubmed and medscape. Then find and print the entire research article (a few scientific journals are available at SMC Library, many more are available at cost online, and most journals are available for free at UCLA’s biomedical library). Your presentation will consist of: - Introduction to the physiology of the system - Analysis of the original research article o Introduction to the question investigated o Methods o Results (include data figures here) o Conclusions o Implications for your classmates - Critique of the journal article o Did their data convince you? Why or why not? o What is the biological significance of the findings? o What other experiments would you like to see? You are welcome to be creative with your presentations. You may use the white board, overhead transparencies, PowerPoint presentations, or any other media that is effective, and that encourages student involvement. Your presentations should be 15 minutes in length, with a 5 minute question/answer session to follow. Make sure that all members of your group contribute equally in the class presentation. Please turn in a sheet of paper detailing each person’s contributions before your presentation. Please email or give me the journal article by April 1. You are welcome to first email the citation or abstract to make sure that it is an appropriate article. If you need any audiovisual equipment that we do not already have in the lab room, please let me know in advance. You will be graded as follows: background physiology (10), methods (10), results (10), conclusions (10), critique (10), quality of presentation (10), encouraging student involvement (10), and participating in other presentations (5), for a total of 75 points. Good luck, and have fun with this! Physiology 3 Lab Manual, Spring 2012 13 Lab 1: Skills Lab Goals: To become comfortable with: 1. Scientific notation 2. Solute concentrations 3. Using a pipetman 4. Calibrating equipment 5. Graphing and the standard curve 6. The scientific method Background: The tools listed above are fundamental to the study of physiology. We will be using each of these tools in future experiments. Familiarity with these concepts is crucial in allowing you to complete and understand the labs in this course. Experiment: You will do these exercises in groups of two. Make sure that each of you practices these techniques and becomes familiar with these tools. Lab Notebook: Answer the following questions in order and in pen in your lab notebook. No formal lab report is required. Leave your notebook in the designated lab box before you leave. Activity 1: Understanding scientific notation, scale, and significant figures 1. Scientific notation The numbers used in physiology are typically very large or very small. For example, the amounts of substances found inside cells are very small yet the numbers of molecules are still quite large. The most convenient way to express this great range of values is by scientific notation, in which numbers are expressed as products of a number times ten to some power. Values less than one and greater than zero are expressed with negative exponents, while values greater than one are expressed with positive exponents: 0.1 = 10-1 0.01 = 10-2 0.001 = 10-3 1 = 100 10 = 101 100 = 102 1000 = 103 For example: the number 367 can be expressed as 3.67 x 102. The number 0.052 can be expressed as 5.2 x 10-2. Physiology 3 Lab Manual, Spring 2012 14 2. Scale 10-9 L nL nanoliter 10-6 L ųL microliter 10-3 L mL milliliter 1L L liter 103 L kL kiloliter 3. Significant figures and rounding off Imagine that you have weighed out 8.8 g of sodium, and that you would like to calculate how many moles of sodium that equals. You divide 8.8 g by sodium’s molecular weight of 58.44g/mol, and your calculator indicates that the result is 0.1505817 moles. This impressive figure incorrectly suggests that you were able to measure the number of moles to the nearest ten-millionth of a mole! In truth, your response cannot be more accurate than the initial reading. If your scale could only measure to an accuracy of the tenths unit, or 0.1 g, then all calculations derived from these measurements can be no more accurate than that. You will have to round off the calculated figure. In our example, the hundredths unit (0.1505817 moles) is no longer a significant figure, so it must be rounded off. If the hundredths unit were a 4 or lower, you would round down the tenths digit to 1 (0.1 moles). If the hundredths unit were a 6 or higher, you would round up the tenths digit to 2 (0.2 moles). However, since the hundredths unit is a 5, you alternately round up and then down. Questions: 1. Convert standard notation to scientific notation: 0.0035 2. Convert scientific notation to standard notation: 5.35 x 10-2 3. 100ųL is the same as how many mL? 4. How many moles are in 10.0 g of sodium? (MW=58.44g/mol) Activity 2: Understanding solute concentrations 1. Molarity When you mix two substances together, you create a solution. In solutions, a solute is dissolved in another substance known as the solvent. For example, if you are making sugar water, the sugar is the solute, and it is added to water, which is the solvent. The concentration of sugar in that solution can be expressed in terms of molarity, which specifies the number Physiology 3 Lab Manual, Spring 2012 15 of moles of solute per liter of solution. A mole is a fixed number of solute molecules: Avogadro’s constant (6.022 x 1023) number of molecules. Molarity (molar, or M) = moles of solute liters of solution Because molarity describes the number of solute molecules in a solution, the actual amount of solvent added to the solute is variable, and depends on the quantity of solute. As you can see in the image below, beaker 2 has more solute molecules, so less solvent than beaker 1, for a 1.0 L solution. Molarity examples: a. If you measure out 1.0 mole of sucrose (342.3 g) and add water until it reaches 1 L, what is the molarity of the resulting solution? 1.0 mole sucrose = 1.0 mol, or 1.0 M sucrose 1.0 L solution L b. If you measure out 2.0 moles of sucrose and add water until the solution reaches the 1.0 L mark, what will the resulting molarity be? 2.0 moles sucrose = 2.0 mol, or 2.0 M sucrose 1.0 L solution L c. If you dissolve 116.88 g NaCl in enough water to make a 2.0 L solution, what is the resulting molarity? For this one you have to convert weight in grams to number of moles.The molecular weight of NaCl is 58.44 g/mol. 116.88 g NaCl x 1 mol = 2.0 mol NaCl 58.44g 2.0 mol NaCl = 1.0 M NaCl 2.0 L solution 2. Molality Physiologists more commonly use molality to measure solute concentration because the ratio of solute to solvent molecules is of critical importance. Molality specifies both the solute and solvent amounts: Molality (molal, or m) = moles of solute kilograms of solvent Physiology 3 Lab Manual, Spring 2012 16 The solvent amount is specified in kilograms, because different solvents have different densities. The solvent you will be using in this class is water, which has a density of 1.0 kg/L. Therefore, if the solvent is water, the above equation can be simplified: molality = moles solute /L water. Later in the course you will also come across the term “osmolality (Osm).” This is used to describe the molality of a solution that has a several different types of solutes, as in a body fluid. Osmolality effectively adds the moles of the various solutes and expresses that as a ratio to the amount of solvent. Molality example: a. If you measure out 1.0 mole of sucrose (342.3 g) and mix it with exactly 1.0 L of water, what is the resulting molality? Density of water = 1.0 kg/L, so 1.0 L weighs 1.0 kg Molality = 1.0 mol sucrose = 1.0 mol = 1.0 m sucrose 1.0 kg water kg 3. Percent solution Solutions can also be expressed as a percent: the weight of solute to the volume of solution. A 1% solution is defined as 1 g of solute per 100 mL of solution. Percent solution example: a. A 4% NaCl solution is made by adding 4 g of NaCl to 100 mL solution. Questions: Show your calculations. Units (M, m, g, L) are required throughout the course. 5. 80.0 g glucose (molecular weight 180 g/mol) is dissolved in enough water to make 1.0 L solution. What is the molarity of the solution? 6. You are planning on making (a) 1.0 L of a 0.5 M solution of sucrose (molecular weight = 342 g/mol) and (b) 1.0 L of a 0.5 M solution of glucose (molecular weight = 180 g/mol). How much of each do you weigh out? 7. In 1.0 L of a 1.0 M aqueous solution, (a) how many moles of solute are there? (b) How much solvent (trick question)? In 1.0 L of a 1.0 m aqueous solution, (c) how many moles of solute are there? (d) How much solvent? 8. What is the resulting molality if 0.75 mol is dissolved in 2.5 L of water? 9. Which solution has more solutes, a 1.0 m solution of glucose or a 1.0 m solution of NaCl? (Hint: NaCl ionizes) 10. If I ask you to make 100 mL of a 2% solution of glucose, how much would you weigh out? Physiology 3 Lab Manual, Spring 2012 17 Activity 3: Working with the Pipetman 1. Acquaint yourself with the range of volumes that can be measured with each of the pipetmen: Pipetman Maximum volume Range P20 .02 mL (20 ųL) 1 – 20 ųL P200 .2 mL (200 ųL) 20 – 200 ųL P1000 1 mL (1000 ųL) 200 - 1000 ųL P5000 5 mL (5000 ųL) 1 - 5 mL 2. To adjust the volume, turn the volume adjustment knob until the digits represent the volume that you need to pull up. The top number should not exceed the first number of the pipetman (2 for P20 and P200; 1 for P1000, and 5 for P5000). For example, 5 mL looks like “500” in a P5000, 1000 ųL looks like “100” in a P1000, and 50 ųL looks like “050” in a P200. 3. Attach a disposable tip by pressing the pipetman firmly into the base of the tip without touching either the pipetman or the tip. Use the correct tip size. 4. Press the plunger gently to the first stop, lower it into the fluid to be dispensed (holding it vertically), and release the plunger slowly, making sure that the fluid slowly enters the tip and does not get the foam filter wet. Then place the tip in the receiving vessel (eg. test tube) and press the plunger again to the first stop. Wait for 2 seconds, until the fluid is dispensed, and then press the plunger farther to the second stop to make sure it is all out. 5. Avoid getting fluid into the pipetman: keep your pipetman vertical at all times, and pull fluid up slowly. 6. The same tip can be used repeatedly in the same reagent, from low to high concentration. Be careful not to contaminate. 7. Remove the tip by gently rotating it and placing it in the appropriate lined waste container or in the trash. Questions: 11. Which is the best pipetman for measuring 100 ųL? 12. If you measure out 500 ųL in a P1000, which numbers do you see in the display, from top to bottom? 13. If you take the P20 and dial “020” from top down, how much fluid are you pulling up? Activity 5: Calibrating equipment Many instruments that measure physiological parameters require calibration first, so that the equipment can determine the relationship between the input and the output response. In the enzyme lab, we will be using a Physiology 3 Lab Manual, Spring 2012 18 spectrophotometer, which measures how much light is absorbed by a solution. The concentration of a solution is directly proportional to the amount of light it absorbs. The opposite of absorbance is transmittance, which is the amount of light that passes through the solution unabsorbed and reaches the other side. The spectrophotometer must be calibrated each time it is used, so that the absorbance value represents only the light that is absorbed by the solution of interest, not by other molecules that may happen to be present. Calibration is accomplished by first inserting a cuvette that has the same composition as the experimental solution, minus the solute of interest. This is called the “blank.” Calibration instructions: 1. Turn on the spectrophotometer, let it warm up for 15 minutes. 2. Set the wavelength to 410nm. 3. Mix the blank tube (in this case, use 5 mL water). 4. Make sure you are measuring “transmittance.” If you have an analogue spectrophotometer, make sure you are looking at the top row. If you have a digital spectrophotometer, make sure to switch the digital view to transmittance. 5. Using the knob on the left (the same one you used to turn on the spectrophotometer) adjust the needle or number to zero transmittance. 6. Place the blank into the spectrophotometer. Using the right knob, adjust the needle or the number to 100% transmittance. You are informing the machine that it should ignore any molecules that it encounters because they are not the solute of interest. 7. The spectrophotometer is now calibrated. 8. When you perform the enzyme lab, you will do this calibration, and then switch to reading absorbance values. You will then place the experimental solutions into the machine and read absorbance values. The spectrophotometer should be recalibrated every hour. Question: 14. Which other solutes you could potentially find in or on your cuvette that would bias the measurement of solute concentration? Activity 6: Graphing and the Standard Curve Graphs are commonly used in physiology to display data: X axis (horizontal line) Y axis (vertical line) Each axis must be labeled with the identity of the variable and the units by which it is measured, and the scale must be linear (1, 2, 3, 4, 5 etc.). Physiology 3 Lab Manual, Spring 2012 19 Slope is a measure of the steepness of a line. A standard curve is a particular type of line graph that relates two measures. We will be making a standard curve for the enzyme lab, and this will relate absorbance from a spectrophotometer and solute concentration. First, several known concentrations of the experimental solute will be mixed and inserted into a calibrated spectrophotometer. Absorbance values will be recorded and a graph will be made. A straight line is drawn that best incorporates all of the points on the graph. Not all of the points will fall on the line. Later, when unknown concentrations of solute are placed in the spectrophotometer, the absorbance readings can be located on the graph and used to determine solute concentration. To do this, locate the absorbance value on the y-axis, travel horizontally to the curve, drop down to the x-axis, and this point is the solute concentration at that absorbance reading. The conversion can also be calculated using the slope of the line. Physiology 3 Lab Manual, Spring 2012 20 Questions: 15. Imagine you have mixed the following known concentrations of a solution. You then measured their absorbances in the spectrophotometer. Construct a standard curve (a line) from the following data points. You must use graph paper and a ruler. Each axis must be labeled with the identity of the variable and its units, and the scale must be linear (1, 2, 3, 4, 5 etc.). Tube Known concentration (M) Absorbance (nm) 1 0 0 2 0.1 0.03 3 0.5 0.06 4 1.0 0.08 5 5.0 0.28 16. If you place a cuvette with an unknown concentration of that solute in the spectrophotometer, and it reads an absorbance of 0.15nm, what is the concentration of solute inside? 17. Now calculate the slope of your line. Show your calculations. 18. Use the slope of your line to calculate the concentration of solute if the absorbance is 0.15nm. Show your calculations. Activity 7: Scientific method The scientific method is a set of techniques that serves to answer scientific questions. It involves the following systematic method of inquiry: Hypothesis: A simple yet specific statement of the effect of the treatment. Stated as: “The [treatment] will [have a specific effect] on [parameter of interest] compared with the [control]. For example: Drinking 16 oz. of Gatorade decreases urine volume compared with drinking the same amount of water. Experiment: The hypothesis is tested by designing and performing an experiment, which often involves testing effects of a treatment. A well- designed experiment will include randomly assigned groups and unbiased measures of the outcome. The experiment often includes the following: a) Treatment: The item you are testing. In the example: drinking Gatorade b) Control: The standard of comparison. All conditions are identical to those of the treatment group, except the treatment itself. In the example above: drinking an equal amount of water. Physiology 3 Lab Manual, Spring 2012 21 c) Dependent Variable: The variable being measured or tested. Example: urine volume Conclusion: Either validation or invalidation of the hypothesis based on the results of the experiment. Analysis of the effect of a treatment centers around statistical significance (are the groups statistically different?), biological significance (is the difference biologically relevant?), and scope of inference (was the experiment only performed on middle-aged Caucasian women?). Repeated verification of a hypothesis may result in a theory. Eventually, the theory may become a law, or principle. Questions: Consider the following experiment (reference below). It has long been thought that antioxidants decrease risk for developing cancer. This was tested by giving either the antioxidant Vitamin E or placebo at random to 35,000 men, and measuring how many were diagnosed with prostate cancer 5 years later. There was no statistically significant difference in prostate cancer rate between the groups at the end of the study. 19. What was their hypothesis? (Be specific) 20. What was their treatment group? 21. What was their control group? 22. What was the dependent variable? 23. Can they broadly conclude that antioxidants do not prevent cancer? Why or why not? Reference: Lippman SM et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers. JAMA. December 9, 2008. Physiology 3 Lab Manual, Spring 2012 22 Lab 2: Homeostasis Goals: 1. To become familiar with writing a lab report 2. To learn how to design and execute a physiology experiment 3. To understand the concept of homeostasis Background: Homeostasis is the ability of a body to regulate its internal environment so as to maintain a stable, constant condition. In this lab session, you will test your ability to maintain homeostasis of a chosen body parameter. Experiment: Pick one body parameter that normally maintains homeostasis and is easy to measure (e.g. body temperature). Take 5 baseline readings of your parameter of interest, all in one sitting, and record this in your lab notebook. This will give you an idea of the precision, or reproducibility, of your recording device (e.g. is your baseline range 98.4°- 98.7°F (precise measurements) or 92.5° - 99.9°F (not precise)). Please note that this will not tell you about the accuracy of your recording device, which is the degree of closeness of your measurement to its true value (e.g. if your thermometer reads 93.8°F when your body temperature really is 98.6°F your device is not accurate). Choose one experiment to challenge homeostatic regulation of the parameter you choose. Be creative! Record your dependent variable before and after your treatment. If you do manage to perturb your homeostasis, please document its return to baseline. Please do not design an experiment that will pose a health risk. Make sure to review the principles of the scientific method on page 20 before designing your experiment. Lab Notebook: You will be writing a formal lab report for this experiment. Use the information on pages 8-11 of this manual to guide you in writing your report. Make sure to include all required sections of the report and to display results using either tables or graphs (use a ruler). Do not forget to include your 5 baseline precision measurements in your data section. Make sure to also answer the questions on the following page. The report can be typed or handwritten, and is due at the enzyme lab. During the enzyme lab, staple or tape this lab report into your lab notebook. Physiology 3 Lab Manual, Spring 2012 23 Questions: 1. What was your hypothesis? 2. What was your treatment? 3. What was your control? 4. What was your dependent variable? 5. How far did your body deviate from homeostasis? 6. If you managed to perturb your homeostasis, how quickly was baseline recovered? 7. Given your data, did your body maintain homeostasis of the parameter you measured? Why, or why not? 8. Given your 5 baseline readings, did the precision of the measuring device affect your experiment? Why, or why not? (i.e. did your dependent variable post treatment fall outside the range of baseline measurements?) 9. Design a large-scale study to address the same hypothesis. You may use a large but realistic sample size. Physiology 3 Lab Manual, Spring 2012 24 Lab 3: Enzymes Goals: 1. To understand the activity of enzymes 2. To determine the effects of substrate concentration, temperature, and pH on enzyme activity Background: Enzymes are biological catalysts that increase the rate of chemical reactions. Trypsin is a pancreatic enzyme that catalyzes the cleavage of peptide bonds, thereby breaking down proteins into smaller proteins or amino acids. Trypsin is active specifically at the peptide bonds that have carboxyl groups donated by the amino acids arginine and lysine. In this experiment, we will use a synthetic arginine-containing peptide substrate called N-benzyl-DL-arginine-p- nitroanilide HCl (BApNA). BApNA will be hydrolyzed by trypsin. When BApNA is hydrolyzed, a yellow substance called p-nitroaniline will be released, and this can be measured colorimetrically, using a spectrophotometer. Today’s experiment involves measuring the amount of BApNA that is being catalyzed by trypsin, and testing which factors affect the activity of trypsin. Experiment: You will carry out these experiments in groups of four. Each group will do the standard curve, and will be assigned one of the other experiments to do. Lab Notebook: 1. Record the names of your group members 2. In your own words, define the following: enzyme, substrate, product, pH, and absorbance 3. Make a standard curve 4. Record data from your experiment in your lab notebook, paying attention to units 5. Post your graph on the board 6. Use the data from your classmates to answer the questions in order Activity 1: Make a p-nitroaniline standard curve 1. Obtain 5 cuvettes and label them 1-4 and B with a wax pencil. 2. Pipette the following into the indicated tube: Tube 10-4 M p- Tris buffer Final molar concentration nitroaniline solution of p-nitroaniline 1 2.50 mL 2.50 mL 5.0 x 10-5 2 0.50 mL 4.50 mL 1.0 x 10-5 3 0.25 mL 4.75 mL 0.5 x 10-5 4 0.05 mL 4.95 mL 0.1 x 10-5 B (blank) 0.00 mL 5.00 mL 0 Physiology 3 Lab Manual, Spring 2012 25 3. Calibrate the Spectronic 20: a. Turn on the spectrophotometer, let it warm up for 15 minutes. b. Set the wavelength to 410nm. c. Mix the blank tube specific to the experiment (tube B). d. Using the knob on the left (the same one you used to turn on the spectrophotometer) adjust the needle to zero transmittance (the zero on the left). e. Place the blank into the spectrophotometer. Using the right knob, adjust the needle to the right, to 100% transmittance. f. The spectrophotometer is now calibrated. Save the blank in the tube rack, in case another calibration is necessary. g. Switch to reading absorbance values; on an analogue spectrophotometer, this just means reading the bottom values. 4. Mix your solutions by shaking the cuvette. Visually confirm that all of the cuvettes have equal volume of solution. 5. Read the absorbance of tubes 1-4. 6. Record your results in a table in your lab notebook. Make sure that your table has the concentration information from the table above. 7. Plot a standard curve of absorbance v. p-nitroaniline concentration. Draw a straight line which best fits the point obtained. Hint: page 19. Experiment 1: Effect of substrate concentration on reaction rate 1. Obtain 6 cuvettes and label them 1-5 and B. 2. Pipette the following into the indicated tubes: Tube 10-3 M BApNA Tris buffer Final molar solution concentration of BApNA 1 5 mL 0 mL 1.0 x 10-3 2 4 mL 1 mL 0.8 x 10-3 3 3 mL 2 mL 0.6 x 10-3 4 2 mL 3 mL 0.4 x 10-3 5 1 mL 4 mL 0.2 x 10-3 B (blank) 2.5 mL 2.5 mL 0.5 x 10-3 3. Visually confirm that all tubes have the same volume of solution. 4. Incubate the tubes for 2 minutes in the 37ºC incubator. 5. To tube B add 0.1 mL of 0.001 M HCl. Mix and wipe the cuvette with a kimwipe. Calibrate the spectrophotometer using tube B as a blank. 6. Add 0.1 mL trypsin enzyme to tubes 1-5 and mix by shaking the tubes. 7. Record the time. Return the cuvettes to the incubator and record absorbance values every 2 minutes for 10 minutes. Make sure to dry the cuvettes each time before measuring absorbance. 8. Convert each absorbance to concentration using the standard curve (see page 19). Physiology 3 Lab Manual, Spring 2012 26 9. Plot end-product concentration v. time for all five curves on one graph. Distinguish the lines using different colors or symbols. Connect the points. Experiment 2: Effect of temperature on enzyme activity 1. Obtain 5 cuvettes and label them 1-5. 2. Pipette 5 mL BApNA into tubes 1-5. Incubate these for 5 minutes at their respective temperatures (transfer the contents of tube 4 to a high test tube tube before placing in hot water): Tube BApNA Temperature 1 5.0 mL Ice water (0-5ºC) 2 5.0 mL Room temperature (22-25ºC) 3 5.0 mL Body temperature (37ºC) 4 5.0 mL Boiling water (100ºC) B (blank) 5.0 mL Room temperature (22-25ºC) 3. Add 0.1 mL of 0.001 M HCl to tube B. Mix and wipe the cuvette with a kimwipe. Calibrate the spectrophotometer using tube B as a blank. 4. Add 0.1 mL trypsin enzyme solution to tubes 1-4 and record the time. 5. Quickly return the cuvettes to their respective temperatures. Allow the cuvettes to incubate for 3 minutes. Then remove a cuvette and wipe dry with a kimwipe. Read the absorbance of the tube and immediately return the cuvette to its incubator. Repeat for the rest of the tubes. 6. Measure absorbance of all tubes every 3 minutes for a total of 9 minutes. 7. Convert absorbance to concentration using the standard curve (page 19). 8. Plot end-product concentration v. time for all four curves on one graph. Distinguish the lines using different colors or symbols. Connect the points. Experiment 3: Effect of pH on enzyme activity 1. Obtain 6 cuvettes and label them 1-5 and B 2. Pipette the following into the indicated tube: Tube BApNA pH buffer solution 1 1 mL 4 mL pH 6 2 1 mL 4 mL pH 7 3 1 mL 4 mL pH 8 4 1 mL 4 mL pH 9 5 1 mL 4 mL pH 10 B (blank) 1 mL 4.0 mL Tris Physiology 3 Lab Manual, Spring 2012 27 3. Wait for 2 minutes 4. To tube B add 0.1 mL of 0.001 M HCl. Mixand wipe the cuvette with a kimwipe. Calibrate the spectrophotometer using tube B as a blank. 5. Add 0.1 mL trypsin solution to tubes 1-5. Record the time and incubate each in the incubator at 37 ºC. At 5 and 10 minutes, read the absorbances of the tubes and record the results. Make sure to wipe the tubes dry with a kimwipe before each spectrophotometer reading. 6. Convert each absorbance to concentration using the standard curve (see page 19). 7. Plot end-product concentration v. pH. Plot two separate lines, one for 5 minute and one for 10 minute readings. Distinguish the lines using different colors or dashed lines. Connect the points. Questions: 1. Which substance is measured by the spectrophotometer in these experiments? 2. What did the standardization curve allow you to calculate? 3. What effect did increasing the substrate concentration have on reaction rate? Why? 4. What effect did temperature have on the reaction rate? Why? 5. What effect did pH have on the reaction rate? Why? Disposal All solutions can be disposed of in designated waste container. Place all pipetman tips in the trash, and all glassware in the soaking bucket in the sink. Physiology 3 Lab Manual, Spring 2012 28 Lab 4: Osmosis Goals: 1. To understand the concept of osmosis 2. To understand the effects of extracellular fluid composition on the cell Background: The cell membrane is made up of phospholipids and is semipermeable. The lipid portion of the membrane is hydrophobic, and the phosphate portion is hydrophilic. In the first experiment, you will investigate the direct effect of solute concentration on the sheep red blood cell. The second experiment will explore the movement of water across an artificial semipermeable membrane by osmosis, and will determine the effect of solute concentration on osmosis. Experiment: You will perform experiments in groups of 3-4. Lab Notebook: 1. Record the names of your group members 2. Define the following: osmosis, osmotic pressure, hypertonic / hypotonic, hydrophilic / hydrophobic 3. Record all data first for activities 2-3 and draw graph for activity 3 4. Answer questions in order Activity 1: Osmolality Osmolality is a measure of the number of solutes in a given amount of solvent. Osmolality (Osm) = osmoles of solute kilograms of solvent If 180 g of glucose and 180 g of fructose are dissolved in the same liter of water (both have molecular weight of 180 g/mol), the osmotic pressure, or the pressure driving water into a solution, would equal that of a 360 g/L glucose solution. Osmolality does not depend on the chemical nature of the solutes, but rather on the number of solutes. The molality of this sugar solution is 2.0 m, but is written as 2.0 Osm, because it contains more than one type of solute. When electrolytes like NaCl ionize in water, the number of solutes, and thus the osmolality, is higher than that of a nonionizing solute (double, for NaCl). Blood plasma osmolality is equal to 0.3 Osm, or 300 mOsm. Solutions that have the same osmolality as plasma are said to be isosmotic (equal osmolality), or isotonic (equal pressure) to plasma. Solutions that have a higher concentration of solute than that of plasma are called hypertonic, and those that have a lower concentration of solute are called hypotonic. Physiology 3 Lab Manual, Spring 2012 29 Questions: 1. Which of the following solutions has the greatest osmotic pressure (i.e. greatest number of moles): 30% sucrose (MW = 342g/mol), 60% sucrose, or 30% magnesium sulfate (MW = 246.4g/mol)? Why? Show your calculations. Magnesium sulfate is ionic. The definition of a percent solution can be found on page 16. 2. Would 30% NaCl have the same, increased, or decreased osmotic pressure as 30% magnesium sulfate? (MW = 58.44g/mol, ionic) Why? 3. Saline infusions are often provided medically to rehydrate individuals. It is only meant to increase blood volume, not to change the ratio of solute to solvent. What osmolality do you think the solution is? 4. Calculate the percent saline (NaCl) solution that would be isotonic. The molecular weight of NaCl is 58.44 g/mol. Show your calculations. Remember that NaCl is ionic. Activity 2: Effect of solute concentration on cell membranes 1. Obtain 5 test tubes. Label them. Put on gloves. 2. Add 2 mL of solution 1 to tube 1, and record the solute content of the solution. Place 2 mL of solution 2 into tube 2, and so on for all 5 tubes. 3. Add one drop of sheep blood to each test tube. Mix well by shaking. 4. Using a transfer pipette, place a drop of solution 3 onto a slide, and cover it with a coverslip. Observe the cells at 400x on the microscope. Do the same for the other solutions and draw your results. Make sure to cover your work area with a paper towel. Questions: 5. Which solution was likely isotonic? 6. Would a cell in a .2% saline solution be in a hyper-, hypo-, or isotonic solution? 7. What would happen to the cell in question #5, and why? Physiology 3 Lab Manual, Spring 2012 30 Activity 3: Osmosis across an artificial semipermeable membrane 1. Three solutions will be prepared for you and placed in an artificial semipermeable membrane (“dialysis tubing”): 30% sucrose, 60% sucrose, and 30% magnesium sulfate (MgSO4). 2. The dialysis tubing will be placed in a beaker of water. 3. Record the height of the column of water every 10 minutes for 60 minutes. 4. Draw one graph of column height over time, representing the different solutions with different colors. Questions: 8. Why did the columns rise? Describe the mechanism. 9. Using your graph, calculate the osmosis rate for each solution (cm/time) at the start. Example: Initial rate: 60.0-0.0 cm = 2.0 cm 30.0 min min 10. Which column rose the most? Why? Was that expected? Why, or why not? 11. The osmosis rate in these set-ups will slow down over time. Why? 12. Is sucrose hydrophilic or hydrophobic? Polar or nonpolar? Disposal Used slides should be placed in the disinfectant bucket. Test tube contents can be dumped into the labeled waste container, and tubes placed in the glass disposal bin. Place used transfer pipettes and soiled gloves in the biohazard container. Clean your work area with disinfectant. Physiology 3 Lab Manual, Spring 2012 31 Lab 5: Neurobiology Goals: 1. To become familiar with reflex circuitry in the nervous system 2. To understand the effect of sensory stimuli on efferent output Background: The autonomic nervous system (ANS) is part of the peripheral nervous system. Its goal is to maintain homeostasis of the visceral organs. In this lab we will examine the mammalian diving reflex, which involves the ANS. In mammals, submerging the face in water initiates several physiological responses that maximize the time that can be spent under water. This includes a reduction of heart rate (bradycardia) and peripheral vasoconstriction. The reflex arc is as follows: the trigeminal nerve (CN5) of your face, nose, and mouth senses the cold temperature of the water, relays this information to the brainstem, which then sends parasympathetic efferent instructions to the heart via vagus nerve (CN10) and sympathetic output to peripheral blood vessels. This mix of sympathetic and parasympathetic responses is unique to diving. In humans, mild bradycardia is also caused by breath-holding without submersion. The physiological goals of the diving reflex are to reduce the energetically costly aerobic activity of the heart, to shunt blood to the vital organs, and to conserve core body heat. Experiment: Work in groups of 3-4. Pick one person to be the diver; the others will measure and record data. The diver should be able to perform a breath hold and head dunk without a significant increase in anxiety. The diver should not have had any stimulants in the hour prior to class (eg. caffeine). The diver may keep his/her finger in the pulse oxymeter for the duration of the experiment. I will provide snorkel and mask, though you are welcome to bring your own if you prefer. Lab Notebook: 1. Record the names of your group members 2. Define the following terms: autonomic nervous system, bradycardia, reflex arc 3. Record all data first 4. Answer questions in order Experiment 1: Effect of breath-holding 1. Baseline: Record resting heart rate. Keep finger in pulsometer. 2. Breath-holding: Subject should hold his/her breath for 45 seconds. Record heart rate at that time. Physiology 3 Lab Manual, Spring 2012 32 Experiment 2: Effect of snorkel 1. Wait for subject’s heart rate to return to normal. 2. Baseline: Place finger in pulsometer and record. 3. Snorkel: Subject should breathe through a snorkel. Record data at 45 seconds. Experiment 3: Effect of facial submersion with snorkel 1. Wait for subject’s heart rate to return to normal. 2. Limited facial submersion with breathing: Subject should put on both snorkel and face mask and immerse face in water. Record data at 45 seconds of immersion. 3. Wait for subject’s heart rate to return to normal. 4. Full facial submersion with breathing: Subject should immerse his/her face in water (no face mask) while breathing through the snorkel. Record data at 45 seconds of immersion. Experiment 4: Effects of facial submersion with breath-holding 1. Wait for subject’s heart rate to return to normal. Place finger in pulsometer. 2. Limited facial submersion with breath-holding: Subject should put on a face mask and submerse face in water while holding breath. Take heart rate measurement at 45 seconds of submersion. 3. Wait for subject’s heart rate to return to normal. 4. Full facial submersion with breath holding: Subject should hold his/her breath and immerse the face in water (without face mask). Take heart rate measurement at 45 seconds of submersion. Experiment 5: Effect of water temperature on the diving reflex 1. Wait for subject’s heart rate to return to normal. Place finger in pulsometer. 2. 35ºC: Immerse the subject’s face without face mask or snorkel in a water bath of approximately 35ºC. Record the results at 45 seconds. Also record the temperature of the bath. 3. Allow several minutes between tests for recovery. 4. 10 ºC: Repeat with ice water (not lower than 10 ºC). Record the heart rate at 45 seconds, and the temperature of the ice bath. 5. Include the data from your previous results at room temperature. 6. Record all temperature data on board (10ºC, 23ºC, and 35ºC). Physiology 3 Lab Manual, Spring 2012 33 Questions: 1. Which two trials would you compare to assess the effect of only breath holding (no submersion)? What was the magnitude of the effect (actual numerical difference in heart rate)? Make sure to only compare one variable. 2. Which two trials would you compare to assess the effect of only facial submersion (without breath-holding)? What was the magnitude of the effect (actual numerical difference in heart rate)? Make sure to only compare one variable. 3. Comparing your answers from questions # 1 and # 2, was the diving response due primarily to breath-holding or facial exposure to water? Explain your answer. 4. For experiment 4, what was the hypothesis? (Make sure it is a statement about the effect of the variable you are testing.) 5. For experiment 4, what was your (a) treatment? (b) control? Remember that your treatment must be directly related to your hypothesis. 6. What was the purpose of the mask? And what was its effect on heart rate? 7. What effect did varying the temperature of the water bath have on the diving reflex? 8. Draw the neural circuit involved in the diving reflex and label the sensory and motor nerves. 9. Why is this reflex arc part of the autonomic nervous system, and not the somatic nervous system? 10. Design a different experiment that tests some aspect of the diving reflex in humans. Make sure to state your hypothesis, control, and treatment groups, and dependent variable. Each student must come up with a unique experiment. (3 points) 11. List at least 2 shortcomings of the experiment you designed for #10. (2 points) Physiology 3 Lab Manual, Spring 2012 34 Lab 6: Sensory Physiology Goals: To become comfortable with the mechanisms underlying sensory physiology and the ways in which some of these sensations are tested. Background: The senses include cutaneous, gustatory, olfaction, vestibular, auditory, and visual senses. Specialized receptors, such as chemoreceptors, mechano- receptors, thermoreceptors, and photoreceptors, sense information about the external world and send neural impulses to the brain. This allows us to interpret the world around us. In today’s lab, you will explore your own sensory physiology and explore the mechanisms underlying each sense. Experiment: Work in teams of 2, and perform each of these tests on yourselves. Record your own information in your lab notebook. Lab Notebook: 1. Name of your partner 2. Record your own data from all of the sections first in your lab notebook 3. Answer questions in order at the end Activity 1: Cutaneous Senses 1. Two-point discrimination Use the calipers to determine the two-point threshold of your partner’s palm, back of the hand, fingertip, and back of the neck. Start with the calipers wide apart and the subject’s eyes closed. Randomly alternate two-point with one-point contact so that your partner can’t anticipate you. The partner tells you whether they feel one or two contacts. Decrease the distance between the needles until you partner can no longer accurately tell you how many points are touching. You partner records this distance, and you proceed to another body part. When you are done, switch roles. Questions: 1. Describe why two points are sometimes felt as two, and sometimes as one. 2. The two-point threshold test assesses size of cutaneous receptive fields. Describe receptive field, as it relates to this test. 3. How does the varied receptive field throughout the body relate to neural representation in the homunculus? Use an example. Physiology 3 Lab Manual, Spring 2012 35 2. Referred pain Use the reflex hammer and gently tap the ulnar nerve where it crosses the medial epicondyle of the elbow. Describe the location where you perceive tingling or pain. Questions: 4. What is the physiological mechanism behind this form of referred pain? 5. How does it differ from pain referred from an organ like your heart? Activity 2: Vision 1. Visual acuity: Snellen eye chart Stand 20 ft. away from the Snellen eye chart. Covering one eye, attempt to read the line with the smallest letters you can see (with glasses off, if you wear contact, you can leave them on). Your partner can determine your visual acuity. Record this in your notebook. Repeat this procedure with the other eye. Questions: 6. If you look at the 20/20 line and it is in focus, where in the eye is the image projected onto? If a person’s eyesight is worse than 20/20, where is that image projected? 7. Describe the physiology underlying sight, from a photon of light striking a rod to the action potential traveling to the brain in cranial nerve 2. 2. Astigmatism Stand 20 ft. away from the astigmatism chart and cover one eye (glasses off). If you have astigmatism, some of the spokes will appear sharp and dark, and others will be blurred and light. Repeat this with the other eye. If you have astigmatism that is corrected with eyeglasses, repeat the test while standing 10ft away and rotating your glasses 90º. The shape of the wheel should change when the glasses are rotated. Record any astigmatism in your notebook. Question: 8. If a person has astigmatism, why are some of the spokes blurred? Physiology 3 Lab Manual, Spring 2012 36 3. Color vision Use the color-blind tests provided, which are a series of colored dots arranged in circles. Look at these. A person with normal vision will see a number embedded within each circle. Question: 9. Why would a color-blind individual see something different? 4. Blind spot Follow the instructions on the blind-spot card. Questions: 10. What is the physiological basis for the blind spot? (Be specific) 11. You don’t normally notice a blind spot when looking out of only one eye – what is it that you see in its place? Why is that? 5. Bleaching Stare at a light bulb from 2 ft. away for 15 seconds, and then suddenly shift your gaze to a white sheet of paper. Question: 12. What is the physiological explanation for this illusion? Please be specific. For 1 minute, stare at the dot on the red paper, keeping your head steady. Then suddenly shift your gaze to a sheet of white paper. Repeat this for the blue square. Question: 13. What is the physiological explanation for this illusion? Please be specific. Hint: white light contains all of the wavelengths of visible light. Activity 3: Hearing 1. Rinne’s test Strike a tuning fork with a rubber mallet or your hand to produce vibrations. Place the handle of the vibrating tuning fork against the mastoid process of the temporal bone, with the tuning fork pointed down and behind the ear. This allows conduction by bone directly to the cochlea. When the sound has almost died away, move the tuning fork (by the handle) near the external auditory meatus. If there is no Physiology 3 Lab Manual, Spring 2012 37 damage to the middle ear, the sound will reappear. Simulate conduction deafness by repeating Rinne’s test with a plug of cotton in your ear. Record your experience. Question: 14. When you simulate conduction deafness, why can you still hear the tuning fork when it is on your mastoid process, but not when it is near your external auditory meatus? 2. Weber’s test Place the handle of the vibrating tuning fork on the midsagittal line of your head and listen. In conduction deafness, the sound will seem louder in your affected ear because it is not competing with outside noises, but in sensory deafness, the sound will be louder in the normal ear. Repeat this test with one ear plugged. On which side is the tuning fork louder? Question: 15. Explain why the sound is louder in the affected ear in conduction deafness, and louder in the normal ear in sensory deafness. Use what you know about the underlying causes of these two pathologies. 16. Describe the process of hearing, from sound waves striking the oval window to the neural signal traveling in cranial nerve 8. 17. Describe how the cochlea processes different frequencies. Activity 4: Vestibular System 1. Vestibular-ocular reflex Have the subject sit in a swivel chair with the eyes open and head flexed forward (chin almost touching chest). Rotate the chair quickly to the right for 20 seconds. (Do not do this if you get sick) Abruptly stop the chair and have the subject open eyes as wide as possible. Note the direction of nystagmus. (Subject should then close eyes until dizziness goes away). Switch subjects and repeat with your head resting on a shoulder (parallel to the floor). Note the direction of the nystagmus. Questions: 18. Describe how the vestibular system is stimulated by the spinning. Physiology 3 Lab Manual, Spring 2012 38 19. Describe the nerves and synapses involved in the vestibular-ocular reflex. 20. Why might the change in head position have changed the direction of the nystagmus? Be specific. Physiology 3 Lab Manual, Spring 2012 39 Lab 7: Article Review Goals: 1. To become familiar with the structure and content of scientific journal articles 2. To learn to critically analyze the data presented in primary source articles Background: We will be discussing and analyzing a scientific research journal article. We will first discuss the underlying physiology, and then explore the paper, one section at a time. Please read the article before coming to class, and print the article to bring to class. It will be posted on ecompanion. At the end of the discussion, you will write a review of the paper. Lab Notebook: Perform a review of this article. You are a research scientist who has been given this article by a journal. The journal editors are considering it for publication, and would like your opinion about whether or not it should be published. This is called “peer review.” Your review should consist of the following sections: 1. A one paragraph summary of the paper, using phrases such as “these authors…” Make sure to briefly mention the authors’ objectives, methods, results, and conclusions. 2. A one paragraph summary of your overall opinion, including whether you believe it should be published. Consider whether these data may be valuable to the scientific community. Support your statements. 3. General comments: a bulleted list of major issues. For example, there may be problems with the study design, study execution, conclusions drawn, experiments missing, etc. For each point, state why it is problematic (how it affects interpretation of the data), and how you recommend that the authors overcome the problem. Make sure to consider the biological significance and scope of inference in order to evaluate whether the resulting conclusions can be reliably drawn from the study. 4. Specific comments: a bulleted list of issues with clarity, organization, graphical presentation, etc., as well as recommendations for how the authors can fix the issue. Please make sure that your review is organized, thoughtful, and neat. Physiology 3 Lab Manual, Spring 2012 40 Lab 8: Digestive System Goals: 1. To understand the digestion of carbohydrates, proteins and lipids in the gastrointestinal tract 2. To understand the conditions under which the enzymes of the digestive system are acting, and how these conditions impact macromolecule digestion Background: Digestion of carbohydrates begins in the mouth, where sugars are mixed with saliva containing the enzyme salivary amylase. This breaks down the polysaccharide starch into the disaccharide maltose. In contrast, the digestion of protein begins in the stomach with the enzyme pepsin. This enzyme breaks large polypeptide chains down into shorter chains, and eventually into amino acids. Last, the breakdown of fats occurs in the small intestine through the action of pancreatic lipase. This breaks triglycerides down into their components: fatty acids and monoglyceride. This process is aided by the presence of bile salts from the liver, which break up the large fat droplets, thereby increasing the surface area available to lipase. In this experiment we will test the enzymes and conditions necessary to digest large macromolecules. Experiment: Perform these experiments in groups of 2-4. Perform all 3 experiments in parallel to save time. Lab Notebook: 1. Record the names of your group members 2. Define the following: digestion, absorption, secretion, amylase, pepsin, lipase, bile, bile salt 3. Record your data first 4. Answer questions in order at the end Experiment 1: Digestion of Carbohydrates 1. Label 4 clean test tubes 1-4. 2. Obtain 9 mL of saliva in a small, graduated cylinder. 3. Add 3.0 mL distilled water to tube 1. 4. Add 3.0 mL saliva to tubes 2 and 3. 5. Add 3 drops of HCl to tube 3. 6. Boil the remaining saliva in a separate Pyrex test tube. Let it cool. When cool, add 3.0 mL of this boiled saliva to tube 4. 7. Add 5.0 mL cooked starch to each of the test tubes. 8. Mix these tubes well by shaking or vortexing. 9. Incubate all tubes for 1.5 hours at 37ºC. Physiology 3 Lab Manual, Spring 2012 41 10. Mix the tubes well again. 11. Divide each sample by pouring half of each into four new test tubes. 12. Test one of the sets of four solutions for starch by adding a few drops of iodine to each tube. A purplish black color indicates presence of starch. 13. Test the other four solutions for monosaccharides and disaccharides by: a. Adding 5.0 mL Benedict’s reagent to each of the four test tubes and immerse them rapidly in boiling water bath for 2 minutes. b. Remove the tubes from the boiling water and rate the amount of simple sugars according to the following scale: i. Blue (no monosaccharides and disaccharides) ii. Green (very little) iii. Yellow (some) iv. Orange (significant) v. Red (a lot of monosaccharides and disaccharides) Questions: 1. Based on your experimental results, what does amylase do? 2. What were the effects of pH changes on the activity of amylase? Why? 3. What was the effect of boiling on the activity of amylase? Why is that? 4. Which of the experimental conditions do you think most closely mirrors what happens in your mouth? Why? Experiment 2: Digestion of Protein 1. Label 4 clean test tubes 1-4. Cut 4 slices of egg white about the size of a fingernail and as thin as possible. They must be uniform in size and ultra thin. Place one slice in each of the 4 test tubes. 2. Add 1 drop of distilled water to tube 1. 3. Add 1 drop of HCl to tubes 2, 3, and 4 4. Add 5.0 mL pepsin to tubes 1 and 2. 5. Add 5.0 mL distilled water to tube 4. 6. Place tubes 1, 2, and 4 at 37ºC. 7. Add 5.0 mL chilled pepsin to tube 3 (place pepsin in ice first), and incubate tube 3 in an ice bath as soon as possible. 8. Incubate all tubes for 1.5 hours, remove the tubes. 9. Record the appearance of the egg white. Questions: 5. Based on your experimental results, what does pepsin do? 6. Under which pH conditions does pepsin work best? Why? 7. Did pepsin act more successfully in ice or at 37ºC? Why? Physiology 3 Lab Manual, Spring 2012 42 Experiment 3: Digestion of Triglycerides 1. Label 4 clean test tubes 1-4. Add 3.0 mL litmus cream to each tube. Cream is rich in neutral triglycerides. Litmus is a pH indicator which is blue in alkaline conditions and red in acidic conditions. 2. Add 6.0 mL distilled water to tube 1. 3. Add 3.0 mL bile extract and 3.0 mL distilled water to tube 2. 4. Add 3.0 mL pancreatic lipase and 3.0 mL distilled water to tube 3. 5. Add 3.0 mL pancreatic lipase and 3.0 mL bile extract to tube 4. 6. Mix the tubes. 7. Incubate these at 37ºC for 1.5 hours. Shake and record any color changes. Questions: 8. Why does pH change? (Bile is neutral; pancreatic juice is basic) 9. Based on your results, which secretions are needed to digest triglycerides? 10. Describe the role that pancreatic lipase has in digestion. 11. Describe the role that bile extract has in digestion. Disposal Benedict’s and litmus solutions should be disposed of in the waste container. All other solutions can be disposed of down the drain. Place all pipetman tips in the trash, and all glassware in the soaking bucket in the sink. Physiology 3 Lab Manual, Spring 2012 43 Lab 9: Blood Goals: 1. To become familiar with the various types of cell found in blood 2. To understand the physiology underlying blood typing Background: Centrifuging blood results in a separation of discrete layers. The top layer consists of plasma, and the bottom layer contains formed elements. We will be performing both a hematocrit and a cell count. We will also practice blood typing, which is an important test for compatibility of blood types, and is based on antigens on the surface of red blood cells. Experiment: Perform these experiments in groups of 3-4. No open-toed shoes permitted. Lab Notebook: 1. Record the names of your group members 2. Define the following: erythrocyte, hematocrit, antigen, antibody, agglutination 3. Record data first 4. Answer questions in order at the end Activity 1: Blood cell count Obtain a slide of blood cells and a microscope. Identify all of the major formed elements in one field of view. Count the number of each type of element in a field of view at the highest objective using a counter. You are welcome to count all cells in a quadrant of the field and multiply by 4. Questions: 1. Which formed element is most numerous? What is its function? 2. Which leukocytes did you see in the blood? What are their functions? 3. What is smallest formed element? What is its function? 4. Where are all of these formed elements made? Activity 2: Hematocrit Put on a pair of gloves. Obtain a capillary tube and place its tip in the sheep blood, allowing the blood to move up the tube by capillary action. Then cap the bottom (blood side) with sealing clay. Place the capillary tube in the Physiology 3 Lab Manual, Spring 2012 44 centrifuge, clay side facing out, along with at least one from another group. The capillary tubes must balance each other on either side of the centrifuge. When you are ready, close the lid and start the centrifuge at the MHCT setting. When it is done, measure the hematocrit with the ruler provided and record this number in your data section. Questions: 5. Normal sheep hematocrit is 30-35%. List two circumstances that could cause the measured sheep hematocrit to be too low (25%). 6. What does the hematocrit number measure? 7. Why is it important? 8. What is on the bottom layer? Middle “buffy coat”? Top? Activity 3: Blood typing You will be using fake blood to ascertain the blood type of 4 fake individuals: 1. Obtain a blood test card. Place a drop of anti-A serum in the circle with that label. Replace the cap (always do this if you are not using the bottle). 2. Place a drop of anti-B serum in its circle. 3. Place a drop of anti-Rh serum in its circle (this tests for Rh factor). 4. Put a drop of blood cells in each circle. 5. Rock the test card back and forth for 1 minute, but keep each mixture in its circle. Tilt the card to drain the mixtures to the side of their circle, but not out of their circles. Look for agglutination and record the blood type of each individual. The Rh reaction may take as long as 5 minutes to take place. Questions: 9. List all of the fake individuals with their blood types. 10. Why did certain mixtures agglutinate? Use specific physiological terms from lecture. 11. How would the mixing of incompatible blood types cause health problems in a human? Disposal Capillary tubes must be disposed of in the sharps container. Soiled gloves must be placed in the biohazard container; those that are not soiled may be thrown away. Fake blood test cards may be thrown in the garbage. Physiology 3 Lab Manual, Spring 2012 45 Lab 10: Cardiovascular System Goals: 1. To become comfortable with the measurement and significance of a variety of ECG values, including specific waves, intervals, and heart rate 2. To understand the physiology underlying blood pressure measures, and to learn how these values change when your body is physiologically challenged 3. To learn the origin of heart sounds and pulse Background: The electrocardiogram (ECG) measures waves of depolarization and repolarization of the cardiac myocytes. As many as 12 different recordings can be taken, each giving a different picture of the function of the heart. In this lab, you will take a resting ECG of your heart using the bipolar limb leads. You will analyze the wave segments of your ECG. Blood pressure is the pressure that blood places on vessel walls, particularly, the systemic arteries, during the systolic and diastolic activities of the heart. In this lab, you will practice using the blood pressure cuff and stethoscope in order to learn the underlying physiology, and then test which values change upon lying down or exercising. Last, you will use a stethoscope to assess heart rate and to listen to the heart sounds. You will challenge your heart rate to determine how quickly it can change. Experiment: Work in groups of 2-4. Do each recording on yourself. We have two ECG machines, so you will have to rotate through these. We have enough blood pressure cuffs and stethoscopes for everyone. Lab Notebook: 1. Record the names of your group members 2. Define the following: cardiac myocytes, pacemaker cells, cardiac systole and diastole, blood pressure systole and diastole 3. Record data from each section first 4. Answer questions in order at the end Physiology 3 Lab Manual, Spring 2012 46 Activity 1: ECG 1. Preparing for the ECG: Clean two areas of skin with an alcohol swab: R radial styloid process and left lower tibia. Attach an electrode sticker to each area, tab facing the heart. Then apply the following leads to these stickers: White – right wrist; Red – left ankle. Check display to make sure that the starting point is Manual Lead II. Locate on the control panel the following buttons: Run/Stop , forward and backward There will be about a 2 second delay before the paper starts to run The Run/stop button is pressed only at the beginning and the end of the series. 2. To record: Press the button once to start, there will be a 2 second delay and then the paper will move. Press Run/Stop button once to Stop Obtain your ECG strip. Paste the strip into your lab notebook under the data section. Note that on the ECG, the x axis represents time (each box is 1 mm, or .04 s) and the y axis represents intensity (0.1 mV per mm) Questions: 1. On your ECG strip, mark the following: a. P, Q, R, S, T waves b. Atrial depolarization c. Ventricular depolarization d. Start to end of ventricular mechanical contraction (systole) e. Start to end of ventricular mechanical relaxation (diastole) You can do this for one cardiac cycle, which is the duration between one event and the next comparable event. For example, the duration between one R wave, and the next R wave of the ECG. Physiology 3 Lab Manual, Spring 2012 47 2. Calculate your heart rate: ________1 beat __________ x __60 seconds__ = __# beats__ X seconds for one cycle 1 minute minute Remember that 1 big box represents .2 seconds. 3. The ECG shows the electrical activities of the heart. How are these electrical waves related in time to the mechanical contraction/relaxation of the heart? 4. Why is the QRS complex so much higher in amplitude than the other waves? 5. Which interval represents the AV nodal delay? Why is that delay important? Activity 2: Blood pressure 1. Have someone record your blood pressure while you are standing, lying down, standing again, and after exercising. To take a manual blood pressure, a sphygmomanometer and stethoscope are needed. Make sure to first clean the stethoscope ear piece with an alcohol wipe (and wipe it down after use as well). Wrap the cuff of the sphygmomanometer snugly around the arm, above the elbow. Place the stethoscope diaphragm under the cuff, over the brachial artery (just medial to the biceps tendon). Inflate the cuff to about 180mm, using the bulb pump. Inflation collapses the brachial artery, stopping blood flow. Deflate the cuff gradually, at about 3mmHg per second. Watch the needle of the display and listen for Korotkoff sounds, the sounds made by the blood as it begins to move again. Systolic pressure is the pressure at which you first hear sounds. Diastolic pressure is the pressure at which you first hear no more sounds. Average blood pressure in a young healthy adult is around 120/80. Make sure that you also become comfortable taking other students’ blood pressure. Questions: 6. Why are there no sounds with the stethoscope when the cuff is inflated to 180 mmHg? 7. What is the basis for the Korotkoff sounds in the middle pressure range? Describe in detail. 8. Why are there no more sounds when the cuff is deflated below 60 mmHg? 9. Why are high and low blood pressure dangerous? Physiology 3 Lab Manual, Spring 2012 48 Activity 3: Heart Sounds 1. Listen and record heart sounds. Place the diaphragm of the stethoscope over your chest, between the 4th and 6th ribs on the left side. Specific sounds may be found in the following locations: 1: Aortic semilunar valve: right second intercostal space 2. Tricuspid Valve: right, fifth intercostal space 3. Bicuspid Valve: left, 6th intercostal space 4. Pulmonary semilunar valve: left, 2nd intercostal space Questions: 10. What causes the lub sound? And the dub sound? 11. In relation to ventricular systole and diastole, when do you hear lub? Dub? Activity 4: Heart Rate 1. Have a partner determine your heart rate by taking a pulse rate. Locate the ulnar or radial artery at the wrist. Gently press against the vessel with index and middle finger, adjusting slightly to locate the pulses. Count the number of pulses that are felt in six seconds. Multiply that number by ten to determine your heart rate (BPM = beats per minute). 2. Next, have your partner do something that will increase heart rate, and measure again. You can exercise, ask an embarrassing or stressful question, etc. Questions: 12. Why do you feel movement on your wrist that correlates with the contractions of your heart? 13. Did heart rate change quickly? What aspect of the nervous system is responsible for this increase? Physiology 3 Lab Manual, Spring 2012 49 Lab 11: Urinary System Goal: To understand how ingestion of various substances affects urine volume Background: The kidneys are important regulators of homeostasis in the body. They regulate pH, electrolyte concentration, and blood volume. Thus, proper kidney function is vital for life. Many mechanisms control urine volume, including the amount of sodium reabsorbed by the nephrons, the amount of antidiuretic hormone released by the posterior pituitary, and activation of atrial natriuretic peptide and the rennin-angiotensin-aldosterone system. In this lab you will be designing and executing an experiment to test the effect of fluid or electrolyte intake on urine volume. Experiment: As a class, you will design an experiment to test the effect of ingested substances (water, caffeine, Gatorade, salt) on your urine volume. You will be assigned to either the control or treatment group. Come to class prepared to perform your experiment. Normal values are included on the next page. In addition, do a urine analysis on 3 samples of fake urine, comparing them with normal values found on the next page. You do not need to record your values, but please confirm your diagnosis with me before the end of class. Lab Notebook: For this lab, you will write a formal lab report, as specified on pages 8-11. It will be due in one week. Questions: 1. What was your hypothesis? 2. What was your treatment? 3. What was your control? 4. What was your dependent variable? 5. What effect did the treatment have on urine volume? 6. What is the mechanism by which the treatment affected the dependent variable? 7. Glucose levels are normally low in urine. Which region of the nephron is responsible for this? By what mechanism is it reabsorbed? 8. Protein levels are normally low in urine. Which region of the nephron is responsible for this? By what mechanism is protein prevented from excretion? Physiology 3 Lab Manual, Spring 2012 50 Dipstick Significance Solute Analysis Glucose Only very small amounts of glucose should be excreted by the kidney (<30 mg/dL) because glucose is reabsorbed in the proximal convoluted tubule. Bilirubin This is a heme breakdown product that is normally reabsorbed in the intestines. Elevated levels (>0.1 mg/dL) indicate hemolysis or liver disease. Ketone Normally, ketones are not detectable in urine (<2 mg/dL). High ketone levels indicate ketoacidosis, starvation, or other metabolic disorder. Specific The normal specific gravity of urine is between 1.001 – 1.035. gravity High and low levels are indicative either of hydration state or pathology. Blood Normally, hemoglobin is not detectable in urine (<0.01 mg/dL) because red blood cells are not filtered in the glomerulus. Presence of blood cells indicates urological, nephrological, or bleeding disorders. pH Normal range is 4.6 – 8. Certain dietary conditions can increase the acidity of the urine, and bacterial growth can increase the alkalinity of the urine. Protein Very little protein is normally present in the urine (<15 mg/dL) because protein is not filtered in the glomerulus and amino acids are reabsorbed in the proximal convoluted tubule. Protein in urine can be the result of kidney disease, but can also be due to strenuous exercise. Nitrite This is normally not detectable in urine. Presence of nitrite >0.075 mg/dL indicates bacterial infection. Urobilinogen This is a breakdown product of hemoglobin and gives urine and feces its color. It is normally present in urine at concentrations up to 1.0 mg/dL. Results >2.0 mg/dL can be due to hemolytic and hepatic disease. Leukocytes Normal clean urine specimens usually yield negative results because cells are not filtered in the glomerulus. An increase in leukocytes (>10/ųL) is indicative of a problem with either the kidney or urinary tract. Physiology 3 Lab Manual, Spring 2012 51 Lab 12: Immune System Goals: 1. To understand the molecular biology of human immunodeficiency virus 2. To become familiar with the enzyme linked immunoabsorbent assay Background: Acquired immune deficiency syndrome (AIDS) is a disease characterized by the progressive deterioration of an individual’s immune system. It is caused by the human immunodeficiency virus (HIV). HIV contains an RNA genome and a reverse transcriptase enzyme. When these are injected into a host white blood cell, they force the host to synthesize HIV DNA and insert it into its own genome. When activated, the host cell will then express HIV genes, resulting in the formation of new HIV particles to be budded out of the cell. HIV is surrounded by a lipid bilayer derived from the host cell membrane during budding. This lipid bilayer contains embedded glycoproteins over a capsid that contains viral proteins. During the early stages of infection, the HIV elicits humoral and cell- mediated responses that result in circulating IgG molecules directed at specific viral antigens. However, the virus has a high mutation rate, and many of the variants survive and escape future immune detection. Enzyme linked immunoabsorbent assay (ELISA) tests can detect specific antibodies or antigens. This HIV ELISA simulation experiment has been designed to detect a hypothetical patient’s circulating IgG’s directed at an HIV antigen. Several wells will first be coated with simulated HIV. Then a simulated sample of human serum will be added to the wells. If the primary IgG is present, it will bind to HIV. Then a secondary antibody is added, which can bind to the primary IgG. This secondary antibody (anti-HIV-IgG) is raised in rabbits and goats and is covalently linked to horseradish peroxidase. When hydrogen peroxide and aminosalicylate are added to each well, the peroxidase converts the peroxide to water and oxygen using salicylate as the hydrogen donor. The oxidized salicylate is brown in color, and indicates a positive result. Experiment: Work in groups of 4. Lab Notebook: 1. Record the names of your group members 2. Define the following: HIV, ELISA, IgG, primary antibody, secondary antibody 3. Record data first 4. Answer questions in order at the end Physiology 3 Lab Manual, Spring 2012 52 Experiment: 1. Obtain a microtiter plate, orient it vertically, and mark the plate with your group name, and number the rows 1-4. 2. Label 5 transfer pipets as: (-), (+), DS1, DS2, PBS. (-) stands for negative, (+) stands for positive, DS stands for donor serum, and PBS stands for phosphate buffered saline. 3. To all 12 wells, add 100µl of “HIV.” Incubate for 5 minutes at room temperature. 4. Remove all the liquid with a fresh transfer pipet. 5. Wash each well once with PBS buffer by taking the PBS pipette, adding PBS to the wells (do not overfill), and then using the same pipette to remove all the liquid from the wells in each row. Dispose of the liquid in the beaker labeled “waste.” 6. Add 100µl of PBS buffer to the 3 wells in row 1 (negative control, no IgG). Add 100µl of the “+” (positive control, IgG, primary antibody) to the 3 wells in row 2. Add 100µl of donor serum “DS1” to the 3 wells in row 3. Add 100µl of “DS2” to the 3 wells in row 4. 7. Incubate at 37°C for 15 minutes. 8. Tell instructor to “make the substrate!” 9. Remove all liquid from each well with the appropriately labeled transfer pipet. Dispose the liquid in the waste container. 10. Wash each well once with PBS buffer as described in step 5. 11. Add 100µl of the anti-HIV-IgG peroxidase conjugate (secondary antibody) to all 12 wells. 12. Incubate at 37°C for 15 minutes. 13. Remove all liquid from each well with the appropriately labeled transfer pipet. Dispose the liquid in the waste container. 14. Wash each well once with PBS buffer as described in step 6. 15. Add 100µl of the substrate to all 12 wells. 16. Incubate at 37°C for 5 minutes. 17. Remove the plate for analysis. If color is not fully developed after 5 minutes, incubate at 37°C for a longer period of time. Disposal Place all waste in labeled container. Microtiter plates may be thrown in the garbage. Questions: 1. What is the role of the primary antibody in this assay? 2. What is the role of the secondary antibody in this assay? Why is it necessary? 3. What does the positive control consist of? Why is it important? 4. What does the negative control consist of? Why is it important? Physiology 3 Lab Manual, Spring 2012 53 5. Why is anti-HIV-IgG screened instead of the virus itself? 6. Describe the entire sequence of immune activation after initial HIV infection. Highlight how it results in formation of HIV IgG’s. (8 points) 7. Why are those IgG’s not enough to conquer an HIV infection? 8. Why does destruction of T helper cells compromise the entire immune system? 9. How would you make a secondary antibody to test for exposure to swine flu (a virus)?
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