Anatomy 1: Human Anatomy - Santa Monica...

Physiology 3 Lab Manual, Spring 2017 1

PhysiologyLaboratory

ManualSpring 2017

Dr. Christina G. von der OheSanta Monica College


Table of ContentsCourse Material Pag

eSyllabus ………………………………………………………………………………………………………

3

Calendar …………………………………………………………………………………………………….

6

Laboratory Safety……………………………………………………………………………………….

7

Instructions for lab notebooks ……………………………………………………………………

8

Instructions for formal lab reports …………………………………………………………….

9

Lab 1: Introductory Lab …………………………………………………………………………….

10

Lab 2: Enzymes ………………………………………………………………………………………….

20

Lab 3: Osmosis …………………………………………………………………………………………..

25

Lab 4: Group Projects………………………………………………………………………………..

28

Lab 5: Neurobiology …………………………………………………………………………………..

29

Lab 6: Sensory Physiology ………………………………………………………………………….

31

Lab 7: Scientific Article …………………………………………………………………………….

35

Lab 8: Digestive System ……………………………………………………………………………

36

Lab 9: Blood …………………………………………………………………………………………….

39

Lab 10: Cardiovascular Physiology …………………………………………………………….

42

Lab 11: Immune System …………………………………………………………………………..

45

Lab 12: Urinary System .…………………………………………………………………………..

48


Physiology 3: Human PhysiologyInstructor: Christina G. von der Ohe, PhD, Professor, Dept. of Life

SciencesOffice: SC-261Phone: (310) 434-4662 Email: [email protected] OH: MW 9:30-11:05 am, TuTh 7:25-7:50am

Meeting Times:

Lecture MW 8:00-9:20am, SC-151Lab section 3101: M 11:15-2:20pm SC-201Lab section 3102: W 11:15-2:20pm SC-201

Student Learning Objectives:

1. Given a problem or set of conditions, write a hypothesis, provide an experimental design, and identify dependent and 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-14th ed. Physiology Laboratory Manual, C. von der Ohe

RequiredMaterials:

Lecture: 4 scantrons #882 or #25110 and a #2 pencil Lab: lined + quad paper, ruler, colored pens or pencils,

calculator

Resources: 2016-17 lecture videos: https://www.3cmediasolutions.org/f/8d5b272d41bf3cca86b4786711b24a90eec6f599 2010 lecture videos: https://www.3cmediasolutions.org/f/3ae85d4e700c8c4f6b3d600568c2976116c796912010 lecture audio: https://www.3cmediasolutions.org/f/f71dfebef69a038f8961b377843070fada90b96c

Attendance:

On-time attendance is recorded. 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

https://www.3cmediasolutions.org/f/f71dfebef69a038f8961b377843070fada90b96c

https://www.3cmediasolutions.org/f/f71dfebef69a038f8961b377843070fada90b96c

https://www.3cmediasolutions.org/f/3ae85d4e700c8c4f6b3d600568c2976116c79691

https://www.3cmediasolutions.org/f/3ae85d4e700c8c4f6b3d600568c2976116c79691

https://www.3cmediasolutions.org/f/8d5b272d41bf3cca86b4786711b24a90eec6f599

https://www.3cmediasolutions.org/f/8d5b272d41bf3cca86b4786711b24a90eec6f599

mailto:[email protected]


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 and suitably documented circumstances, 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.


Lecture: We will start the lecture with an extra credit 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. You are required to read the chapter or listen to the lecture BEFORE class.

Opening Question:

At the start of every session you will answer a brief extra credit question based on the previous or current lecture. 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 and complete the pre-lab assignment before coming to lab. The pre-lab will be turned in at the start of lab. Lab assignments must be individual and unique work and turned in by 2:15pm. Bring your textbook, lab manual, lined and quad paper, ruler, and calculator to each session. There are no make-ups for labs, but I will drop your lowest score.

Class Environment:

I strive to make the classroom a safe and encouraging learning environment. 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 silence cell phones and refrain from texting during class. Food, drink (including water), and gum are not permitted in any of the science rooms.

Presentation:

There will be group presentations on a recent scientific discovery, to be discussed later in the course.

Grading: You will be evaluated based on performance on exams, lab assignments, 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% = A1 final exam 140 80-89% = B11 lab assignments (1 dropped)

250 70-79% = C

Group presentation 75 60-69% = DAttendance 35 Below 60% = FTOTAL POINTS 800

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.


You are responsible for checking grade accuracy throughout the semester. You will not have access to your tests after the final exam.


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 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 Viewing:

We will review the exam in lab. The exams are my property, 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 this class:

Physiology 3 is a very rigorous class that requires considerable discipline, time, and dedication. Tips for success:1. Be prepared for lecture (read chapter or listen to lecture

ahead of time)2. Study the material daily; be sure you can explain

mechanisms from memory3. Be prepared for labs (read lab and complete pre-lab

ahead, bring textbook, and come prepared for data collection)

4. Visit office hours5. Engage in class by asking questions

Academic Dishonesty:

You must do your own work on all opening questions, exams, and lab assignments. 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.

Ask Questions!:

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.


DATE TOPIC FOX CHAPTER

LAB

Feb 13 Introduction & Homeostasis 1 Introductory LabFeb 15 Chemistry 2 Introductory LabFeb 20 HOLIDAYFeb 22 Cell Biology 3 No labFeb 27 Enzymes 4 Enzyme LabMar 1 Cellular Respiration 5 Enzyme LabMar 6 Transport Mechanisms 6 Osmosis LabMar 8 Muscle, Review 12 Osmosis LabMar 13 Lecture Exam 1 Group Projects,

StatisticsMar 15 Intro to Neurobiology 7 Group Projects,

StatisticsMar 20 Autonomic Nervous System 9 ANS LabMar 22 Central Nervous System 8 ANS LabMar 27 Sensory Physiology 10 Senses LabMar 29 Endocrinology 11 Senses LabApr 3 Reproductive System 1 20 Article Review LabApr 5 Reproductive System 2 20 Article Review LabApr 10 SPRING BREAKApr 12 SPRING BREAKApr 17 Digestive System 18 Digestive LabApr 19 Growth + Metabolism,

Review19 Digestive Lab

Apr 24 Lecture Exam 2 No labApr 26 Cardiovascular System 1 13 Blood LabMay 1 Cardiovascular System 2 13 Blood LabMay 3 Cardiovascular System 3 14 Cardiovascular LabMay 8 Immune System 15 Cardiovascular LabMay 10 Respiratory System 1 16 Immune LabMay 15 Respiratory System 2 16 Immune LabMay 17 Urinary System 1 17 PresentationsMay 22 Urinary System 2 17 PresentationsMay 24 Review Urinary Lab: ALLMay 29 HOLIDAYMay 31 Lecture Exam 3 DiscussionJune 5 Review Discussion


June 7 Final Exam 8-11am


Laboratory Safety Rules for Biology, Botany, Physiology, and Zoology

The following is a list of rules that is designed to ensure your safety as well as the safety of your classmates and instructor. Failure to follow these safety rules may result in loss of points from the related assignments. It can also result in the removal from class on the day of the incident and one subsequent class session. In addition, violation of the rules may result in a referral to the Office of Student Judicial Affairs for disciplinary action.

1. Chemicals used in this course are believed to be safe when used according to standard laboratory safety practices. However, their total or long term effects on the human body are not known. The list of chemicals known to cause cancer or reproductive harm is published yearly. Please see your instructor to determine whether you will be exposed to one or more of these chemicals during this course.

2. The effects of chemicals used in this course on human pregnancy are unknown, and pregnant women are advised to consult their physician before taking this course.

3. Eating, drinking, chewing gum, smoking, and the use of cosmetics are not permitted in the laboratory. This includes drinking water. Keep your fingers, pencils, and other objects out of your mouth.

4. Appropriate clothing, including shirt and closed-toe shoes must be worn at all times. Lab coats are required at all times in the Biology 21 and 22 labs. If gloves are required, dispose of the gloves before leaving the room. In some courses, you may be required to wear goggles for specific laboratory experiments. Your instructor will give advanced notice of the dates when those experiments are scheduled.

5. Working in the laboratory without the instructor present is prohibited.6. Keep your personal items out of your work space and aisles at all times.7. If animals or preserved specimens are being used in the laboratory, do not handle them without the

permission and supervision of your instructor, and treat them with respect.8. Do not eat any laboratory specimens or products. 9. Use proper technique in handling containers of acids, bases, and other chemicals. If in doubt, ask your

instructor. Do not pipette by mouth. Note safety signs in the lab, and read precautions in the lab manual or handouts.

10. Follow instructions regarding the use and care of scalpels, scissors and sharp pointed metal probes.11. Follow your instructor’s directions for proper care of laboratory equipment. When plugging equipment

into a receptacle, make sure that your cord does not cross a walkway.12. After working with chemicals at your lab table, wash the table tops with water and soap, and dry them.

Wash your hands with soap and water. 13. Follow the instructor’s directions regarding cleanup and disposal of all lab materials. Make sure that

biohazards, sharps, broken glass, chemicals, and garbage are all disposed of properly. 14. If you break a thermometer or any other glassware, avoid contact with sharp objects and notify your

instructor immediately.15. If any chemicals are spilled on your skin or clothing, flush the affected area with water and notify your

instructor immediately.16. If you splash any substance into your eyes, rinse your eyes for 15 minutes with the eye wash fountain,

notify your instructor, and be seen immediately in the Health Services Office on campus.17. If your clothing catches on fire or you spill any corrosive chemicals on your skin or clothing, use the

emergency shower for 15 minutes, inform your instructor, and be seen in the Health Services Office on campus.

18. If it becomes necessary to evacuate the lab, go immediately to the lawn near the clock tower using the nearest stairway and/or walkway. Stay off of paved areas, parking lots, and other potential fire lanes.

19. Photography, videos, and recordings can only be taken with instructor consent. They may not be given, sold, or published in print or online without the written consent of the instructor, and can only be used

for the purposes of the class.


Physiology Lab InstructionsIn this course, you will be performing experiments and recording and analyzing your data. These activities are designed to improve your skills in laboratory techniques, data analysis, statistical analysis, use of the scientific method, critical analysis of scientific articles, writing, and presenting.

To do before lab:

You are required to read the lab manual and complete the entire pre-lab assignment prior to the start of lab. The pre-lab assignment can be found under the “Pre-lab” heading for each lab. The pre-labs are worth 5-10 points and must be turned in by the start of lab. You are welcome to ask me questions about the pre-lab as you work on it. Definitions must be in your own words.

To bring to lab:

You must bring your lab manual, textbook, lined and graph paper, ruler, calculator, and colored pens or pencils to each lab session.

Lab assignments:

Your lab assignments will be written on 8.5 x 11” lined paper. Use quad paper and a ruler for all graphs and tables. All labs must be written in pen, and presented in the order instructed. You do not need to restate the question. You may bullet point your answers instead of using complete sentences, unless you are writing in paragraph format. You must always include a data section. Typed lab assignments are acceptable as well.

General lab instructions:

Every lab has unique instructions for the lab assignment, which can be found under the “General Instructions” heading. Make sure you follow these instructions, and write up your lab in order. Make sure you include data from each section of the lab (worth 2 points per section!). The labs will be performed in groups, but your written work must be individual and unique. Any shared or copied entries will receive a zero grade. Lab assignments are due by 2:15 of each lab session.

Reviewing your lab:

At the end of each lab period, after your lab work is complete and handed in, you may view the answer key. 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 assignment, or sharing answers with other students will be considered a violation of the Student Honor Code, and will be handled accordingly.


Grading: Your labs will be graded regularly during the semester. Any work that is not affixed to your lab will not be graded.

Formal Lab ReportsIn this course, you will be instructed to write 2 formal lab reports: one for the nervous system lab, and one for the urinary system. These should be well-written reports, with complete sentences and correct grammar, spelling, and punctuation. Paragraphs must have a topic sentence, several supporting sentences, and a concluding sentence. Follow this format for each formal lab report:

1. Name and Date: (and group members for the ANS lab)2. Title: Make sure that the title is descriptive. 3. Introduction: A detailed 1-3 paragraph explanation of the physiology

being explored in the lab. Include your hypothesis for the experiment and the related mechanism. Use complete sentences.

4. Procedure: Briefly list the steps that were actually taken. List statements in past tense. Do not use personal pronouns.

5. Data: This section contains the raw data results of the experiment as well as statistics (mean, standard deviation, p-value). It should also include a graph of means with standard deviation bars. The graph must be labeled, and must be made with a ruler or a computer.

6. Discussion: A detailed 1-3 paragraph explaining the results. What do your observations tell you? What do your statistics indicate? 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 and how could they have affected the data?

7. Questions: Answer any questions posed in the lab manual.


Lab 1: Introductory Lab

Goals: To become comfortable with:1. Scientific notation, scale, and significant figures2. Solute concentrations3. The scientific method4. Using a pipetman5. Making a standard curve

Background:The concepts listed above are fundamental to the study of physiology, and will be used in experiments in this class. Familiarity with these concepts is crucial to allowing you to complete and understand the labs in this course.

General Instructions:No pre-lab, this week only. However, it is wise to work through as much of this lab as possible beforehand. Answer the questions in order.

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.


2. Scale

10-9 L nL nanoliter10-6 L ųL microliter10-3 L mL milliliter1 L L liter103 L kL kiloliter

3. Precision, significant figures, and rounding off

Imagine that you have weighed out 88.8 g of NaCl, and that you would like to calculate how many moles of NaCl that equals. You divide 88.8 g by NaCl’s molecular weight of 58.44g/mol, and your calculator indicates that the result is 1.5195071 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 precise than the initial reading. If your scale could only measure to a precision of three significant figures, then all calculations derived from these measurements can be no more precise than that. The significant figures of a number are those digits that carry meaning contributing to its precision. This includes all digits except leading and trailing zeros (all those before and after the non-zero digits).

In the example above, the calculated figure will need to be rounded off to three significant figures. The thousandths unit (1.5195071 moles) is no longer a significant figure. If the thousandths unit were a 4 or lower, you would round down (1.51 moles). Since the thousandths unit is a 6 or higher, you would round up the hundredths digit to 2 (1.52 moles). If the thousandths unit were a 5, you would alternately round up and then down.

Questions:

1. Convert standard notation to scientific notation: 0.00352. 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 chloride?

(MW=58.44g/mol)

Activity 2: Understanding solute concentrations

1. Molarity

http://en.wikipedia.org/wiki/Accuracy_and_precision

http://en.wikipedia.org/wiki/Numerical_digit


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 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 NaCl2.0 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

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 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 kgMolality = 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 volume of solution. A 1% solution is defined as 1 g solute per 100 mL 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 1.0 L of a 0.5 M solution of sucrose (molecular weight = 342 g/mol). How much sucrose do you weigh out?

7. You are planning on making 1.0 L of a 0.5 M solution of glucose (molecular weight = 180 g/mol). How much glucose do you weigh out?


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 1000ml of a 2% solution of glucose, how much would you weigh out?

Activity 3: 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. HOW TO WRITE A HYPOTHESIS: “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

Gatoradeb) 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.

c) Dependent Variable: The variable being measured. Ex: 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.

11. What was their hypothesis? (Be specific)12. What was their treatment group?13. What was their control group?14. What was the dependent variable?15. Can they broadly conclude that ALL antioxidants do not

prevent ALL cancers? Why or why not? **Careful, this is tricky.Reference: Lippman SM et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers. JAMA. December 9, 2008.

Activity 4: Working with a Pipetman

You will frequently be using a pipetman in lab to measure exact volumes of solutions. We have four sizes of pipetman in this lab, each with a range of volumes that it can measure. Acquaint yourself with these volume ranges:

Pipetman Maximum volume RangeP20 .02 mL (20 ųL) 1 – 20 ųLP200 .2 mL (200 ųL) 20 – 200 ųLP1000 1 mL (1000 ųL) 200 - 1000 ųLP5000 5 mL (5000 ųL) 1 - 5 mL


There is a dial on each pipetman, which you will turn to set the volume in microliters. Each volume display has three numbers in it. If you were to dial the pipetman to maximum volume (for example, to 200ul in the P200), the topmost number in the display corresponds with the first digit (2), the second with the second digit (0), and the third with the third digit (0). Familiarize yourself with the display and volumes of

each of the four types of pipetman:

Pipetman Instructions:1. 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.

2. 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 (the tip color should match the color on the top of the pipetman).

3. 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.

4. Avoid getting fluid into the pipetman: keep your pipetman vertical at all times, and pull fluid up slowly.

5. The same tip can be used repeatedly in the same reagent, from low to high concentration. Be careful not to contaminate.

6. Remove the tip by gently rotating it and placing it in the appropriate lined waste container or in the trash.

Questions:

16. Which is the best pipetman for measuring 100 ųL?17. If you take the P20 and dial “020” from top down, how

much fluid are you pulling up?

Activity 5: Making a standard curve

In the upcoming enzyme lab, you will be measuring the concentration of product made by an enzyme. This current activity will give you the skills needed for successful completion of the enzyme lab.

In order to measure the concentration of a solute, we will use a spectrophotometer, which is a machine that measures how much light is absorbed by a solution (in “absorbance units” or AU). Greater concentrations of solution absorb more light. The opposite of absorbance is transmittance, which is the amount of light that passes through the solution unabsorbed and reaches the other side (“transmittance” (%T)). We will be using a spectrophotometer to measure the concentration of product (in our case: p-nitroaniline) that is made during a chemical reaction.

In order to understand the relationship between absorbance units and concentration of product, you will construct a standard curve. You will place known concentrations of product into the spectrophotometer and record absorbance readings. You will graph these points and make a best fit line (called a “standard curve”) that goes through the origin.

This will allow you to calculate the concentration of product for any absorbance reading.

Here is an example of a standard curve. Note that the axes are linear (equal spacing between the numbers), that the line goes


through 0,0, and that the line makes sure to fall between all of the points rather than going through one particular point. In a future experiment, when unknown concentrations of solute are placed in the spectrophotometer, the slope of this standard curve can be used to relate absorbance readings to solute concentration. The slope is calculated by picking a point on the line, not one of your data points. The second point is the origin (0,0). Divide the y coordinate by the x coordinate. So in this case:

.3 AU = .1 AU/Mx10-5

3 Mx10-5

You will now make a standard curve for p-nitroaniline in groups of 3-5 students. P-nitroaniline is a product made by the enzyme trypsin. Make a photo of this standard curve and your slope calculation because you will be using it in Lab 2.

1. Turn on the spectrophotometer, let it warm up for 15 minutes.2. Obtain 6 cuvettes and label them 1-5 and B with a wax pencil.3. Pipette the following into the indicated tube:

Tube 10-4 M p-nitroaniline

Tris buffer solution

Final molar concentration of p-nitroaniline

1 2.50 mL 2.50 mL 5.0 x 10-5

2 2.0 mL 3.0 mL 4.0 x 10-5

3 1.5 mL 3.5 mL 3.0 x 10-5

4 1.0 mL 4.0 mL 2.0 x 10-5

5 0.5 mL 4.5 mL 1.0 x 10-5

B (blank)

0.00 mL 5.00 mL 0

4. Calibrate the Spectronic 20:a. Set the wavelength to 410nm.b. Using the knob on the left (the one you used to turn on the

spectrophotometer) adjust the needle to zero transmittance.

c. Place the blank into the spectrophotometer. Using the right knob, adjust the needle 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. Calibration complete.

5. Mix your solutions with the micro vortex. Visually confirm that all of the cuvettes have equal volume of solution.


6. Place each tube (1-5) in the spectrophotometer and read the absorbance units (absorbance units are in the bottom row). Check that the data trend makes sense before you continue. No values should be zero.

7. Record your results in a table in your lab notebook. Make sure that your table has the concentration information from the table above (see example at the end of this lab). (2 points)

8. Plot a standard curve of absorbance v. p-nitroaniline concentration (5 points) (see example at the end of this lab). This will tell you the relationship between absorbance and concentration. Draw a straight line that best fits the points and goes through the origin. The line does not have to actually go through any of the points, but it must be an average of all of the information. Your standard curve will be turned in today. Make a copy or photo of this graph because you will need it for the pre-lab next week.

9. Calculate the slope of this line (1 point) (see example at the end of this lab). Make sure to show your units and show your work. Make sure that your slope is as accurate as possible. Your slope will likely be slightly different from your neighbor’s slope. This slope needs to be turned in with your lab assignment today. Make a copy or photo of this slope because you will need it for the next lab.

Disposal

All solutions can be disposed of in the designated waste container. Place all pipetman tips in the trash, and all glassware in the soaking bucket in the sink.


Example Standard Curve and Slope:

Standard Curve Example data:

Tube Final molar concentration of p-nitroaniline

Absorbance Units (AU)

1 5.0 x 10-5 .482 4.0 x 10-5 .393 3.0 x 10-5 .314 2.0 x 10-5 .185 1.0 x 10-5 .08B (blank)

0 0

Standard Curve

Slope of this line*: __.1AU_______ = .1 AU/Mx10-5

1 x 10-5 M* The exact number of your slope will depend on your standard curve. It is calculated based on any point on the line, not one of your data points. It is easiest to keep Mx10-5 as part of your units, so that you can just divide each AU data point by your slope, and all resulting


concentrations will have the units Mx10-5. For a more detailed explanation, see Activity 5.


Lab 2: Enzyme LabGoals:

1. To understand the activity of enzymes2. 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.

General Instructions:You will carry out these experiments in groups of 3-4. Each group will be assigned one of the experiments to do. Record all data and make a graph for the experiment you conduct. Pay attention to units throughout. Use graph paper and a ruler for tables and graphs. Answer the questions neatly and in order at the end of your lab.

Pre-lab: (9 points) In your own words, define the following: enzyme, substrate,

product, pH, absorbance, and transmittance Answer the following questions:

1. Which chemical are you measuring with the spectrophotometer? 2. What will the slope of the standard curve allow you to calculate?3. Write down the slope that you calculated in Lab 1. Use this slope

to calculate the concentration of solute if the absorbance is 0.15 AU. Show your calculations.


Experiment 1: Effect of substrate concentration on reaction rate

1. Obtain 5 cuvettes and label them 1-4 and B.2. Pipette the following into the indicated tubes:

Tube 10-3 M BApNA Tris buffer solution

Final molar 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

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. Place 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 1-4 and mix by shaking the tubes.

7. Record the time. Return the cuvettes to the incubator and record absorbance values every 3 minutes for a total of 9 minutes in a table format. Make sure to dry and mix the cuvettes each time before measuring absorbance. Also make sure that absorbance readings go down as tube number goes up, that all readings are non-zero, and that all the absorbance readings uniformly go up with time.

8. Convert each absorbance to concentration using the slope of your standard curve (see example at end of this lab). Record the concentrations in a table.

9. Plot end-product concentration v. time for all four curves on one graph. Distinguish the lines using different colors. Connect the points.

Experiment 2: Effect of temperature on enzyme activity

1. Obtain 5 cuvettes and label them 1-4 and B.2. Place 1 ml trypsin in another cuvette and place it in boiling water 3. Pipette 5 mL BApNA into tubes 1-4 and B. Place these for 5

minutes at their respective temperatures. From this point on, keep these tubes at their respective temperatures as much as possible.


Tube BApNA Temperature1 5.0 mL Ice (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)

4. 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.

5. Add 0.1 mL of boiled trypsin to tube 4. 6. Add 0.1 mL room temperature trypsin enzyme solution to tubes

1-3 at the same time as step 4. Record the time.7. Immediately return the cuvettes to their respective temperatures

(your goal is to keep them at their temperature for the entire experiment). Wait for 3 minutes. Then remove a cuvette, mix it, 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.

8. Measure absorbance of all tubes every 3 minutes for a total of 9 minutes and record these data in a table. Make sure that you mix and dry each tube before measuring. Make sure that all absorbance readings are non-zero, and go up with time.

9. Convert absorbance to concentration using the slope of your standard curve (see end of this lab for example calculations). Record the concentrations in a table.

10. Plot end-product concentration v. time for all four curves on one graph. Distinguish the lines using different colors. Connect the points.

Experiment 3: Effect of pH on enzyme activity

1. Obtain 6 cuvettes and label them 1-5 and B2. Pipette the following into the indicated tube:

Tube BApNA pH buffer solution Deionized water

1 0.9 mL 1.0 mL pH 6 3.0 mL2 0.9 mL 1.0 mL pH 7 3.0 mL3 0.9 mL 1.0 mL pH 8 3.0 mL4 0.9 mL 1.0 mL pH 9 3.0 mL5 0.9 mL 1.0 mL pH 10 3.0 mLB (blank) 0.9 mL 4.1 mL


3. Wait for 2 minutes4. Mix and 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

place each in the incubator at 37 ºC. 6. At 5 and 10 minutes, read the absorbances of the tubes and

record the results in a table. Make sure to mix and wipe the tubes dry with a kimwipe before each spectrophotometer reading. Make sure that all absorbance readings are non-zero, and that they go up with time.

7. Convert each absorbance to concentration using the slope of your standard curve (see example at the end of this lab). Record these concentrations in a table.

8. 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. Why did increasing the substrate concentration increase reaction rate? Do not restate the question (ex: “Because high substrate concentration increases reaction rate”).

2. Why did reaction rate increase with temperature up until body temperature? Do not restate the question (ex: “Because reaction rate increases with temperature until body temperature”).

3. Why did reaction rate decrease towards boiling? 4. Why was the pH optimum basic? Think about where this enzyme

normally functions.5. Why does the enzyme not function well at pHs that are lower

than the optimum? (don’t restate the question)6. Pepsin is a stomach enzyme that breaks down proteins into

amino acids. List its optimum temperature, pH, and protein concentration (high or low – hint: protein is the substrate).

7. Describe an experiment that you could do to test another factor affecting this enzyme. Make sure to include the treatment, control, dependent variable, and how you would avoid bias.

Disposal


All solutions can be disposed of in the designated waste container. Place all pipetman tips in the trash, and all glassware in the soaking bucket in the sink.


Example Enzyme Experiment Tables, Calculations, and Graphs:

Experiment 1, 2, or 3Example data:

Tube AU at 2 minutes AU at 4 minutes 1 .2 .32 .25 .353 .3 .44 .35 .45

Convert each AU into concentration Mx10-5 (divide each AU by your slope):

Tube Mx10-5 at 2 min Mx10-5 at 4 min 1 2.0 3.02 2.5 3.53 3.0 4.04 3.5 4.5

Now graph the concentration of your product (put units on the axes):


Lab 3: Osmosis LabGoals:

1. To understand the concept of osmosis2. 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.

General Instructions:You will perform experiments in groups of 3-4. Record all data and draw a graph for Activity 2. Answer the questions in order at the end of the lab.

Pre-lab: (8 points) Define the following: osmosis, osmotic pressure, hypertonic /

hypotonic, hydrophilic / hydrophobic Using the information about osmolality below, answer the following

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 in Lab 1.) (2 points)

2. Does 30% NaCl (MW = 58.44g/mol, ionic) have the same, increased, or decreased osmotic pressure as 30% magnesium sulfate? 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 is the osmolality of the solution?

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.

Activity 1: 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:

1. Which solution was likely isotonic?2. Based on your observations, would a cell in a .2% saline solution

be in a hyper-, hypo-, or isotonic solution?3. What would happen to the cell in question #2, and why?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.) (2 points)


Activity 2: 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 above the second

string in cm every 10 minutes for 60 minutes.4. Draw one graph of column height over time, representing the

different solutions with different colors.Questions:

5. Why did the columns rise? Describe the mechanism.6. Using your graph, calculate the osmosis rate for each solution

(cm/time) at the start. Example:

Initial rate: 20.0cm = 2.0 cm 10.0 mi min

7. Which column rose the most? Why? Was that expected? Why, or why not?

8. What if 30% urea (MW=60.06g/mol; nonionic) had been in the dialysis bag instead of 30% magnesium sulfate. Would it have risen higher or lower or the same? Why?

9. The osmosis rate in these set-ups will slow down over time. Why? (They will not become isosmotic because solutes will never leave the bag)


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.


Lab 4: Group PresentationThere is no pre-lab for this. 8% of your grade is based on a group project. You will work in groups of 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. Today you will be assigned your group and topic, and we will go over statistics for physiology.

Your group will be assigned a specific system on which your presentation must be focused. Find a news article on that system that summarizes an original research article from the past year. Track down the original research article using google or pubmed. Send an abstract of the original research article to me for approval. Then download the pdf of the research article and give me both the pdf and a printed copy of your article. Most of these journals are available at cost online as well as for free at UCLA’s biomedical library.

Your presentation will consist of:- Introduction to the physiology of the system *only what is

needed to understand your presentation- Analysis of the original research article

o Introduction to the question investigatedo Methods o Results (include data figures here, point out important

numbers)o Conclusionso Implications for your classmates

- Critique of the journal articleo 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 me a pdf of the journal article and a paper copy before the 8th week of class. You are welcome to first email the citation or abstract to make sure that it is an appropriate article.


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.

Lab 5: Neurobiology LabGoals:

1. To become familiar with reflex circuitry in the nervous system2. To understand the effect of sensory stimuli on efferent output3. To become familiar with designing, executing, and

communicating a scientific research project

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). 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). 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.

General Instructions:Work in groups of 4-6. Come prepared to dive your face; all students must participate. Please limit your stimulant use in the hour prior to class (eg. caffeine). Design an experiment to test the diving reflex. At your disposal you have pulse oxymeters (to measure heart rate), snorkel masks, snorkels, buckets, and different temperatures of water. All individuals in your group will dive twice, once for the control and once for the treatment (alternate the order and rest in between dives). Dive lengths are 40 seconds long. If one or more members of your group cannot hold their breath for 40 seconds, all member of the group should use a snorkel. Record the heart rate at the 40 second mark, immediately before you tell the student that time is up. Write up one formal lab report for your group. Follow instructions for writing a formal lab report found near the start of this manual. You are welcome to analyze the statistics together with me on the class computer.

Pre-lab: (5 points)


Define the following terms: autonomic nervous system, bradycardia, reflex arc

Answer the following questions:1. Describe the two nerves involved in the reflex arc that you are

testing today.2. Describe the neurotransmitters and receptors used by this two-

neuron efferent pathway (both at the ganglion and at the target).


Questions:

1. Why was replication (having a sample size larger than 1) beneficial to your experiment?

2. If one of your classmates drank caffeine before taking part in the experiment, it would introduce bias. Which kind of bias would it introduce, and why is that problematic for data analysis? (We covered this in statistics; see summary below)

3. If every member of your group performed the treatment condition first and the control condition second, it would introduce bias. Which kind of bias would it introduce, and how does that affect data analysis?

Bias: There are two main types of bias. Random bias is due to sampling variability and is quite normal (eg. you are testing the effect of exercise on heart rate, but subjects have varying levels of fitness to begin with). This is can be addressed by statistical analysis and is usually not a serious concern. It manifests as an increase in variability, which makes it more difficult to see differences between groups. Systemic bias is far more lethal to a study; this is caused by a factor that affects one group differently than the other (eg. you are testing the effect of exercise on heart rate and place elderly in the exercise group and teenagers in the non-exercise group). With this type of bias, you are no longer able to determine the effect of the treatment, because the effects are confounded by another variable.


Lab 6: Sensory Physiology Lab

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.

General Instructions:Work in teams of 2, and perform each of these tests on yourselves. Record your own data in your lab and answer questions in order at the end.

Pre-lab: (9 points) Define the following terms: sensory receptor, receptive field, retina,

cochlea, semicircular canal Answer the following:

1. Describe why two cutaneous points can be sometimes felt as two, and sometimes as one.

2. Does light striking a photoreceptor result in its depolarization or hyperpolarization? Why?

3. Describe how a hair cell is bent upon hearing sound.4. Describe how the vestibular system is stimulated by spinning.


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.

Question:

1. Which body part had the smallest receptive field, and how do you think that correlates with the size of its neural representation in the homunculus?

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.

Question:

2. Describe the two mechanisms for referred pain discussed in class. Which of the two is at work in this example?

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.

Question:

3. If you looked at the 20/20 line and it was in focus, where in the eye was the image projected onto? If a person’s eyesight is worse than 20/20, where is that image projected?


2. 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:

4. Why would a color-blind individual see something different?

3. Blind spot Follow the instructions on the blind-spot card.

Question:

5. What is the physiological basis for the blind spot? (Be specific)

4. Bleaching 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:

6. What is the physiological explanation for this illusion? Be specific. Hint: white light contains all of the wavelengths of visible light.

Activity 3: Hearing

1. 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:

7. 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.


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).

Question:

8. How does spinning result in nystagmus?


Lab 7: Scientific ArticleGoals:

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

General Instructions:We will be analyzing a scientific research article (both the PowerPoint and pdf will be posted). Please read the article before coming to class, and print and bring the article to class. At the end of the discussion, you will write a critique of the paper.

Pre-lab: (5 points) Read the assigned article Write a one paragraph summary of the study. This should be well-

written, with a topic sentence, supporting statements, a conclusion, and correct grammar, punctuation, and spelling.

Prepare a rough draft list of strengths and weaknesses of this article (this will be turned in with the pre-lab so make a copy if you need it)

Paper Critique Instructions

Write well-written paragraphs in which you critically analyze this research article. Address biological significance, scope of inference, study design, and whether the conclusions drawn are valid. Make sure that your review is organized, thoughtful, and neat. Your essay should include the following sections:

1. A paragraph discussing its major strengths. Support each statement. (7 points)

2. A paragraph discussing its major weaknesses. For each weakness, support your comments. (7 points)

3. A final paragraph giving your overall opinion of this article as a scientific work. (6 points)


Lab 8: Digestive SystemGoals:1. To understand the digestion of carbohydrates, proteins and lipids in

the gastrointestinal tract2. 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.

General Instructions:Perform these experiments in groups of 3-4. Perform all 3 experiments in parallel to save time. Record all data and answer questions in order at the end. Remember that your lab assignments must be unique and individual work.

Pre-lab: (10 points) Define the following: digestion, hydrolysis, absorption, secretion,

bile, bile salt, emulsification Answer the following questions:

1. What are the actions and optimal pH and temperature for salivary amylase?

2. What are the actions and optimal pH and temperature for pepsin?

3. What are the actions and optimal pH and temperature for pancreatic lipase?


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 for 5

minutes. 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 hour at 37ºC.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. What were the effects of adding HCl on amylase activity? Why?2. What was the effect of boiling on the activity of amylase? Why is

that?3. 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 44. 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 place tube 3 in an ice bath as soon as possible.8. Leave all tubes for 1 hour, remove the tubes.9. Record the appearance of the egg white.

Questions:

4. Under which pH conditions did pepsin work best? Why? Do not restate the question (ex: “Because that is the pH that it works best in”).

5. Did pepsin act more successfully in ice or at 37ºC? Why? Do not restate the question (ex: “Because that is the temperature that it works best at”).

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 hour. Shake and record any color

changes.

Questions:

6. Based on your results, which secretions are needed to digest triglycerides?

7. Describe the role that bile extract has in digestion.8. In the triglyceride experiment, why does the post-digestion pH

decrease? (FYI: bile is neutral and pancreatic juice is basic)9. What do you think the resulting color would have been in tube 4

if you had performed the experiment on ice? Why?


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.


Lab 9: Blood Lab

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.

General Instructions:Perform these experiments in groups of 3-4. No open-toed shoes permitted. Record all data and answer questions in order at the end.

Pre-lab: (10 points) Define: erythrocyte, leukocyte, thrombocyte, plasma, antigen,

antibody, agglutination Answer the following questions:

1. What are the three categories of formed elements and for each, briefly what is its function?

2. What does the hematocrit measure, and why is it important?3. How would the mixing of incompatible blood types cause health

problems?


Activity 1: Blood cell count

Obtain a slide of blood cells and a microscope. Look at blood at 400x magnification and identify all of the three major formed elements in one field of view. Count the number of each type of element in a field of view using a counter. Specifically name and count the leukocytes that you see. 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?2. Which kinds of leukocytes did you see, and what are their roles?3. What does it mean when leukocyte count is high?4. What is smallest formed element?

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 centrifuge, clay side facing out, along with at least one from another group so that they are balanced. 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%. State one health-related circumstance that could cause the sheep hematocrit to be too low.

6. State one experimenter error that could cause the measured sheep hematocrit to be too low.

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 (A antibodies) 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 (B antibodies) in its circle.3. Place a drop of anti-Rh (Rh antibodies) in its circle.4. Put a drop of blood cells in each circle.5. Rock the test card 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. Place the cards on a white paper. Look for agglutination and record the blood type of each individual. The Rh reaction may take as long as 5 minutes to take place.

6. In your data, show which combinations agglutinated.

Questions:

7. List all of the fake individuals with their blood types. (2 points)8. Why did certain mixtures agglutinate? Use specific physiological

terms from lecture.

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.


Lab 10: Cardiovascular SystemGoals:

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 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 assess pulse rate and challenge it to see if it changes.

General Instructions:Work in groups of 2-4. Do each recording on yourself. Record your data from each section and then answer the questions in order at the end.

Pre-lab: (10 points) Define the following: cardiac myocytes, pacemaker cells, cardiac

systole/ diastole, blood pressure systole/ diastole Answer the following questions:

1. Sketch out an ECG. Underneath that, sketch out the atrial and ventricular myocardial action potentials as they relate to the waves of the ECG. Underneath that, sketch out atrial and ventricular contraction, as they correspond in time with the ECG. Your end result should be one figure. (2 points)

2. Why is the QRS complex so much higher in amplitude than the other waves?

3. Which interval represents the AV nodal delay? Why is that delay important?


4. How do the Korotkoff sounds differ from lub/dub in terms of what generates the sounds?

5. Why are high and low blood pressure dangerous?Activity 1: ECG

All students will record their own ECG. Turn on the machine. Clean both wrists and both ankles with an alcohol swab. Attach an electrode sticker to each area, tab pointing down. Then apply the following leads to these stickers: White – right wrist; Green – right ankle; Black – left wrist; Red – left ankle. Make sure that the snap tips are not touching the sticky part of the electrode.

Select the ‘(I, II, III)’ lead channel using the blue up and down arrows. To start the ECG recording, press the ‘MANUAL PRINT’ button. Press the red ‘STOP’ button to stop. You only need a few inches of recording.

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 small box is 1 mm, or .04 s) and the y axis represents intensity (0.1 mV per mm).

Questions:

1. On the Lead II section of your ECG strip, mark the following: (3 points)

a. P, Q, R, S, T waves for 1 cardiac cycleb. Atrial depolarizationc. Ventricular depolarizationd. 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.

2. Calculate your heart rate:

________1 beat __________ x __60 seconds__ = __# beats__ X seconds for one cycle 1 minute minute

1 big box represents .2 sec, and each little box represents .04 sec.


Activity 2: Blood pressure

Each student will have their blood pressure measured. Have someone record your blood pressure while you are sitting, lying down, 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:

3. Why are there no sounds with the stethoscope when the cuff is inflated to 180 mmHg?

4. What is the basis for the Korotkoff sounds in the middle pressure range? Describe in detail.

5. Why are there no more sounds when the cuff is deflated below 60 mmHg?

Activity 3: Pulse

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:

6. What are you actually feeling when you measure your pulse?


7. Describe the mechanism responsible for the rapid rise in heart rate. (neurotransmitters, receptors, channels)


Lab 11: Immune System

Goals:1. To understand the molecular biology of human

immunodeficiency virus2. 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. The host cell will then express HIV genes, resulting in the formation of new HIV particles that bud out of the cell.

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 IgGs directed at an HIV antigen (anti-HIV IgG). Several wells will first be coated with simulated HIV. Then a simulated sample of human plasma will be added to the wells. If the primary antibody 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.

General Instructions:Work in groups of 4. Record your data and answer questions in order.

Pre-lab: (10 points) Define the following: ELISA, immunoglobulin, IgG, primary antibody,

secondary antibody


Answer the following: 1. Describe the entire sequence of immune activation after viral

infection, from being engulfed by an antigen-presenting cell, all the way to formation of anti-viral IgGs. Use the information from lecture (the “putting it together” slide). (5 points)

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 plasma, and PBS stands for phosphate buffered saline.

3. To all 12 wells, add 100µl of “HIV.” Leave the wells 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 labeled pipettes 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, anti-HIV IgG, primary antibody) to the 3 wells in row 2. Add 100µl of donor plasma “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

5.15. 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.

Questions:


1. Is this assay testing for the presence of HIV or the presence of primary antibody?

2. What is the role of the primary antibody in this assay?3. What is the role of the secondary antibody in this assay? Why is

it necessary?4. What does the positive control consist of? Why is it important?5. What does the negative control consist of? Why is it important?6. Why does destruction of T helper cells compromise the entire

immune system?7. Why is it so hard to make a vaccine for HIV?8. Why is it hard to find a cure for HIV?9. How would you develop a secondary antibody for an ELISA for

swine flu (a virus)?

DisposalPlace all waste in labeled container. Microtiter plates may be thrown in the garbage.


Lab 12: Urinary SystemGoal: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 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 water and Gatorade intake on urine volume.

General Instructions: There is no pre-lab this week. The entire class will perform this lab on the day designated in the syllabus. For those of you enrolled in a different lab section: if you cannot attend, you may use the data emailed to you. For all students, the formal lab report is due by 8am on the next day that the class meets.

As a class, you will perform an experiment to test the effect of 20 oz. Gatorade v. 20 oz. water on your urine volume. You will be assigned to either the control or treatment group. Come to class prepared to perform your experiment by bringing your assigned drink and adhering to the protocol below. You will write a formal lab report, as specified near the start of this lab manual. Make sure to follow all of the instructions for the lab report.

Protocol: Before your 8am class, eat a low-salt breakfast and drink 8oz. of

water No substantial eating or drinking between 9-11:15 am 11:15 meet in lab 11:20 urinate and drink assigned fluid (20 oz = 591.4 ml) Make a graduated cylinder by pouring 50ml water into a cup and

marking its height, and repeating that process until you reach the top of the cup

12:30 urinate, measure, dispose of urine in bathroom, and wash hands


Questions:

1. What was your hypothesis?2. What was your treatment group?3. What was your control group?4. What was your dependent variable?5. What is the mechanism by which Gatorade affected the

dependent variable?6. Why does Gatorade have both salt and sugar in it?7. If some students didn’t follow the protocol, which kind of bias

would this create? What is negative about this kind of bias (be specific)?

8. If I assigned students who are fasting to the water group and McDonalds fans to the Gatorade group, which kind of bias would I be creating? What is negative about this kind of bias?

CONGRATULATIONS, YOU HAVE FINISHED ALL OF THE LABS!!!

Anatomy 1: Human Anatomy - Santa Monica...

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