Docstoc

Physiology_Lab_Manual

Document Sample
Physiology_Lab_Manual Powered By Docstoc
					Physiology 3 Lab Manual, Fall 2012    1




                   Physiology
                   Laboratory
                     Manual
                 Fall 2012
       Dr. Christina G. von der Ohe
           Santa Monica College
Physiology 3 Lab Manual, Fall 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, Fall 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: vonderohe_christina@smc.edu
                 OH: MW 7:30-9:00am, 2:30-3:00pm

Meeting          Lecture MW 9:30-10:50am SC-159
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 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.
Physiology 3 Lab Manual, Fall 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, Fall 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 at least 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, Fall 2012                                     6



DATE       TOPIC                        FOX CHAPTER   LAB
Aug 27     Introduction & Homeostasis   1             Skills Lab
Aug 29     Chemistry                    2             Skills Lab
Sept 3     HOLIDAY
Sept 5     Cell Biology                 3             No lab
Sept 10    Enzymes                      4             Enzymes
Sept 12    Cellular Respiration         5             Enzymes
Sept 17    Transport Mechanisms         6             Osmosis
Sept 19    Muscle                       12            Osmosis
Sept 24    Lecture Exam 1                             Group Projects
Sept 26    Intro to Neurobiology        7             Group Projects
Oct 1      Autonomic Nervous System     9             Nervous System
Oct 3      Central Nervous System       8             Nervous System
Oct 8      Sensory Physiology           10            Senses
Oct 10     Endocrinology                11            Senses
Oct 15     Reproductive System 1        20            Article Review
Oct 17     Reproductive System 2        20            Article Review
Oct 22     Digestive System             18            Digestive
Oct 24     Growth and Metabolism        19            Digestive
Oct 29     Lecture Exam 2                             Blood
Oct 31     Cardiovascular System 1      13            Blood
Nov 5      Cardiovascular System 2      13            Cardiovascular
Nov 7      Cardiovascular System 3      14            Cardiovascular
Nov 12     HOLIDAY
Nov 14     Respiratory System 1         16            Presentations
Nov 19     Respiratory System 2         16            Presentations
Nov 21     Urinary System 1             17            Urinary System
Nov 26     Urinary System 2             17            Urinary System
Nov 28     Immune System                15            Immune
Dec 3      Review                                     Immune
Dec 5      Lecture Exam 3                             Discussion
Dec 10     Review                                     Discussion
Dec 17     Final Exam 8-11am
Physiology 3 Lab Manual, Fall 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, Fall 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 as well
   as any statistics. It should also include a table and a graph. The graph 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, Fall 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, Fall 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, Fall 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, Fall 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 October 5. 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, Fall 2012                                              13


                               Lab 1: Skills Lab

Goals: To become comfortable with:
   1. Scientific notation
   2. Solute concentrations
   3. The scientific method
   4. Using a pipetman
   5. Calibrating equipment
   6. Graphing and the standard curve

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, Fall 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. Precision, 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 precise
   than the initial reading. If your scale could only measure to a precision of
   the tenths unit, or 0.1 g, then all calculations derived from these
   measurements can be no more accurate than that. The significant figures of
   a number are those digits that carry meaning contributing to its precision.
   This includes all digits except leading zeros (all those before the non-zero
   digits).

   In the example above, the calculated figure will need to be rounded off.
   The hundredths unit (0.1505817 moles) is no longer a significant figure. 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
Physiology 3 Lab Manual, Fall 2012                                            15


   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 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.
Physiology 3 Lab Manual, Fall 2012                                                 16


   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 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. Which solution is more concentrated: 1.0 L of a 1.0 M aqueous solution,
      or 1.0 L of a 1.0 m solution?
   8. What is the resulting molality if 0.75 mol is dissolved in 2.5 L of water?
Physiology 3 Lab Manual, Fall 2012                                              17


   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?


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.
   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.
   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.

   11. What was their hypothesis? (Be specific)
   12. What was their treatment group?
Physiology 3 Lab Manual, Fall 2012                                             18


   13. What was their control group?
   14. What was the dependent variable?
   15. 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.


Activity 4: 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:

   16. Which is the best pipetman for measuring 100 ųL?
   17. If you measure out 500 ųL in a P1000, which numbers do you see in the
       display, from top to bottom?
Physiology 3 Lab Manual, Fall 2012                                             19


   18. If you take the P20 and dial “020” from top down, how much fluid are
       you pulling up?


Activity 5: Calibrating a spectrophotometer

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
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 (“absorbance units” (AU)). 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)).

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:

   19. Which other solutes you could potentially find in or on your cuvette that
       would bias the measurement of solute concentration?
Physiology 3 Lab Manual, Fall 2012                                             20


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.).




Slope is a measure of the
steepness of a line.




A standard curve (actually, a
line) 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, Fall 2012                                                21


Questions:

   20. 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

   21. If you place a cuvette with an unknown concentration of that solute in
       the spectrophotometer, and it reads an absorbance of 0.15 AU, what is
       the concentration of solute inside?
   22. Now calculate the slope of your line. Show your calculations.
   23. Use the slope of your line to calculate the concentration of solute if the
       absorbance is 0.15 AU. Show your calculations.
Physiology 3 Lab Manual, Fall 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, Fall 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, Fall 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, Fall 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. Also make sure that
      absorbance readings go down as tube number goes up, and that all the
      absorbance readings uniformly go up with time.
Physiology 3 Lab Manual, Fall 2012                                          26


   8. Convert each absorbance to concentration using the standard curve (see
      page 20-21).
   9. Plot end-product concentration v. time for all five 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 1-5.
   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 (pages 20-
      21).
   8. 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 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, Fall 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
      pages 20-21).
   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, Fall 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, Fall 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. 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?
   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 #6, and why?
Physiology 3 Lab Manual, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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, Fall 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 (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 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, Fall 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, Fall 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, Fall 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 sitting, lying
      down, standing, 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, Fall 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, Fall 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 Gatorade 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. Make sure to include a data graph that shows means
and standard deviations for the two groups.

Questions:

   1. What was your hypothesis?
   2. What was your treatment?
   3. What was your control?
   4. What was your dependent variable?
   5. What is the mechanism by which the treatment affected the dependent
      variable?
   6. Glucose levels are normally low in urine. Which region of the nephron is
      responsible for this? By what mechanism is it reabsorbed?
   7. 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, Fall 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, Fall 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 plasma 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, Fall 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 plasma, 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 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.

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, Fall 2012                                                53


   5. Why is the primary antibody 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)?

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:2
posted:9/6/2012
language:Unknown
pages:53