Physiology 3 Lab Manual, Spring 2016
Physiology
Laboratory
Manual
Spring 2016
Dr. Christina G. von der Ohe
Santa Monica College
1
Physiology 3 Lab Manual, Spring 2016
2
Table of Contents
Course Material
Page
Syllabus ………………………………………………………………………………………………………
3
Calendar …………………………………………………………………………………………………….
6
Laboratory Safety……………………………………………………………………………………….
7
Instructions for lab notebooks ……………………………………………………………………
9
Instructions for formal lab reports …………………………………………………………….
10
Lab 1: Introductory Lab …………………………………………………………………………….
11
Lab 2: Enzymes ………………………………………………………………………………………….
20
Lab 3: Osmosis …………………………………………………………………………………………..
26
Lab 4: Group Projects………………………………………………………………………………..
29
Lab 5: Neurobiology …………………………………………………………………………………..
30
Lab 6: Sensory Physiology ………………………………………………………………………….
32
Lab 7: Scientific Article …………………………………………………………………………….
36
Lab 8: Digestive System ……………………………………………………………………………
37
Lab 9: Blood …………………………………………………………………………………………….
40
Lab 10: Cardiovascular Physiology …………………………………………………………….
43
Lab 11: Immune System …………………………………………………………………………..
46
Lab 12: Urinary System .…………………………………………………………………………..
49
Physiology 3 Lab Manual, Spring 2016
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 9:30-11:05 am, TuTh 7:25-7:50am
Meeting
Times:
Lecture MW 8:00-9:20am, SC-151
Lab section 3097: M 11:15-2:20pm SC-201
Lab section 3098: W 11:15-2:20pm SC-201
Student
Learning
Objectives:
1. Given a problem or set of conditions, write a hypothesis,
provide an experimental design, and identify dependent and
independent variables, and control and experimental groups.
2. Identify the physiological mechanisms that each body system
employs to maintain homeostasis.
3. Students should demonstrate confidence in their understanding
of biological concepts and the scientific method to evaluate and
critique current media or a scientific report.
Textbook:
Human Physiology, S. Fox, 10th-14th ed.
Physiology Laboratory Manual, C. von der Ohe
Required
Materials:
Lecture: 4 scantrons #882 or #25110 and a #2 pencil
Lab: lined paper, quad paper, ruler, colored pens or pencils,
calculator
Resources:
Learning Resource Center and Computer Lab
Recordings posted by prior student (out of kindness, so please only
contact her with thanks): http://www.kspill.com/smc/
Homepage: http://homepage.smc.edu/vonderohe_christina/
Attendance:
On-time attendance is recorded. Students who are absent for two
consecutive meetings or to the first exam without informing the
professor with a valid excuse will be dropped from the roster.
Drop Dates:
Drop dates are listed in your catalog. You are responsible for your
enrollment status and the dates and deadlines on 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, Spring 2016
4
Lecture:
We will start the lecture with an extra credit question. The
lecture will be based on PowerPoint slides that will be posted to
ecompanion before lecture. You are welcome to print the slides
before class and take notes on them. You are required to read the
chapter or listen to the lecture BEFORE class.
Opening
Question:
At the start of every session you will answer a brief extra credit
question based on the previous or current lecture. They must be
your own work.
Lab:
In lab, you will design, execute, and record experiments in groups.
You are required to read the lab manual and complete the pre-lab
assignment before coming to lab. The pre-lab will be turned in at
the start of lab. Lab reports must be individual and unique work
and turned in by 2:15pm. Bring your textbook, lab manual, lined
and quad paper, ruler, and calculator to each session. There are
no make-ups for labs, but I will drop your lowest score.
Class
Environment:
I strive to make the classroom a safe and encouraging learning
environment. There will be a lot of class discussion and group
work. Please be respectful of each other. I encourage you to
freely ask questions so that everyone can benefit from the
discussion. This class is for you. Please silence cell phones and
refrain from texting during class. Food, drink (including water),
and gum are not permitted in any of the science rooms.
Presentation:
There will be group presentations on a recent scientific discovery,
to be discussed later in the course.
Grading:
You will be evaluated based on performance on exams, lab
reports, a group presentation, and attendance and participation.
Points will be totaled and expressed as a percent. Grades are nonnegotiable and must be earned.
3 lecture exams
300
90-100% = A
1 final exam
140
80-89% = B
11 lab reports (1 dropped)
250
70-79% = C
Group presentation
75
60-69% = D
Attendance
35
Below 60% = F
TOTAL POINTS
800
At the end, your lecture exam scores will be averaged and your
lowest score will be replaced by the average. Missed exams do not
qualify for this.
Physiology 3 Lab Manual, Spring 2016
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 is cumulative. All books,
notes, and electronic devices must be put away before each exam.
Use the restroom before the exam; you may not leave the room
until you are finished with your exam.
Exam
Viewing:
We will review the exam in lab. The exams are my property, and
may not leave the classroom in any form. I will return your exam
to you and post the answers at the side of the classroom. You will
have 15 minutes to review your exam and ask questions. You may
not take notes, photograph, or leave the room with the exam. Any
of these offenses will result in the filing of an Academic
Dishonesty report and a loss of points on the exam. If you need
more time to review the exam, you are welcome to view it in my
office.
To succeed in
this class:
Physiology 3 is a very rigorous class that requires considerable
discipline, time, and dedication. Tips for success:
1. Be prepared for lecture (read chapter or listen to lecture
ahead of time)
2. Study the material daily; be sure you can explain mechanisms
from memory
3. Be prepared for labs (read lab and complete pre-lab ahead,
bring textbook, and come prepared for data collection)
4. Visit office hours
5. Engage in class by asking questions
I value:
1.
2.
3.
4.
5.
6.
7.
Academic
Dishonesty:
You must do your own work on all opening questions, exams, 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.
Interest in the material
Hard work
Respect for everyone in the classroom
Integrity in your work
Responsibility for your grade
Punctuality
Professional Behavior
6
Physiology 3 Lab Manual, Spring 2016
DATE
Feb 17
Feb 22
Feb 24
Feb 29
Mar 2
Mar 7
Mar 9
Mar 14
Mar 16
Mar 21
Mar 23
Mar 28
Mar 30
Apr 4
Apr 6
Apr 11
Apr 13
TOPIC
Introduction & Homeostasis
Chemistry
Cell Biology
Enzymes
Cellular Respiration
Transport Mechanisms
Muscle, Review
Lecture Exam 1
Intro to Neurobiology
Autonomic Nervous System
Central Nervous System
Sensory Physiology
Endocrinology
Reproductive System 1
Reproductive System 2
SPRING BREAK
SPRING BREAK
FOX CHAPTER LAB
1
No Lab
2
Introductory Lab
3
Introductory Lab
4
Enzyme Lab
5
Enzyme Lab
6
Osmosis Lab
12
Osmosis Lab
Group Projects, Statistics
7
Group Projects, Statistics
9
ANS Lab
8
ANS Lab
10
Senses Lab
11
Senses Lab
20
Article Review Lab
20
Article Review Lab
Apr 18
Apr 20
Apr 25
Apr 27
May 2
May 4
May 9
May 11
May 16
May 18
May 23
May 25
May 30
Jun 1
Jun 6
Jun 8
Digestive System
Growth + Metabolism, Review
Lecture Exam 2
Cardiovascular System 1
Cardiovascular System 2
Cardiovascular System 3
Immune System
Respiratory System 1
Respiratory System 2
Urinary System 1
Urinary System 2
Review
HOLIDAY
Lecture Exam 3
Review
Final Exam 8-11am
18
19
13
13
14
15
16
16
17
17
Digestive Lab
Digestive Lab
No lab
Blood Lab
Blood Lab
Cardiovascular Lab
Cardiovascular Lab
Immune Lab
Immune Lab
Presentations
Presentations
Urinary Lab: ALL
Discussion
Discussion
Physiology 3 Lab Manual, Spring 2016
7
Laboratory Safety Rules for Physiology
The following is a list of safety rules that is designed to ensure your safety as
well as the safety of your classmates and instructor. Failure to follow these
safety rules may result in loss of points or dismissal from the laboratory without
credit, and continued safety issues may result in you being dropped from the
class and receiving a failing grade.
1. Chemicals used in this course are believed to be safe when used according
to standard laboratory safety practices. However, their total or long term
effects on the human body are not known. The list of chemicals known to
cause cancer or reproductive harm is published yearly. Please see your
instructor to determine whether you will be exposed to one or more of
these chemicals during this course.
2. The effects of chemicals used in this course on human pregnancy are
unknown, and pregnant women are advised to consult their physician before
taking this course.
3. Eating, drinking, chewing gum, smoking, and the use of cosmetics are not
permitted in the laboratory. This includes drinking water. Keep your
fingers, pencils, and other objects out of your mouth.
4. Appropriate clothing, including shirt and closed-toe shoes must be worn at
all times. Lab coats are required at all times in the Biology 21 and 22 labs.
If gloves are required, dispose of the gloves before leaving the room.
5. In some courses, you may be required to wear goggles for specific
laboratory experiments. Your instructor will give advanced notice of the
dates when those experiments are scheduled.
6. Keep your personal items out of your work space and aisles at all times.
7. If animals or preserved specimens are being used in the laboratory, do not
handle them without the permission and supervision of your instructor.
8. Do not eat laboratory specimens. Although your instructor may show you a
plant that has edible qualities, it may be contaminated with pesticides or
other toxic chemicals.
9. Use proper technique in handling containers of acids, bases, and other
chemicals. If in doubt, ask your instructor. Note safety signs in the lab, and
read precautions in the lab manual or handouts.
10. Do not pipette by mouth. Use the pipetting bulbs or other instrument for
pipetting as instructed.
11. Follow instructions regarding the use and care of scalpels, scissors and sharp
pointed metal probes.
12. Follow your instructor’s directions for proper care of laboratory equipment.
When plugging equipment into a receptacle, make sure that your cord does
not cross a walkway.
13. Working in the laboratory without the instructor present is prohibited.
Physiology 3 Lab Manual, Spring 2016
8
14. After working with chemicals at your lab table, wash the table tops with
water and soap, and dry them. Wash your hands with soap and water.
15. Follow the instructor’s directions regarding disposal of all lab materials.
Make sure that biohazards, sharps, broken glass, chemicals, and garbage are
all disposed of properly.
16. Follow the instructor’s directions regarding placement of used glassware.
17. If you break a thermometer or any other glassware, avoid contact with
sharp objects and notify your instructor immediately.
18. If any chemicals are spilled on your skin or clothing, flush the affected area
with water and notify your instructor immediately.
19. If you splash any substance into your eyes, rinse your eyes for 15 minutes
with the eye wash fountain, notify your instructor, and be seen immediately
in the Health Services Office on campus.
20. If your clothing catches on fire or you spill any corrosive chemicals on your
skin or clothing, use the emergency shower for 15 minutes, inform your
instructor, and be seen in the Health Services Office on campus.
21. If it becomes necessary to evacuate the lab, go immediately to the lawn
near the clock tower using the nearest stairway and/or walkway. Stay off
of paved areas, parking lots, and other potential fire lanes.
Physiology 3 Lab Manual, Spring 2016
9
Physiology Lab Instructions
In this course, you will be performing experiments and recording and analyzing
your data in lab reports. The pre-lab must be turned in by the start of lab. The
remainder of your lab report will be written during the lab period and must be
turned in by 2:15 of each lab session.
To do before
lab:
You are required to read the lab manual and complete the
entire pre-lab assignment prior to the start of lab. The pre-lab
assignment can be found under the “Pre-lab” heading for each
lab. The pre-labs are worth 5-10 points and must be turned in
by the start of lab. You are welcome to ask me questions about
the pre-lab as you work on it. Definitions must be in your own
words.
To bring to
lab:
You must bring your lab manual, textbook, lined and graph
paper, ruler, calculator, and colored pens or pencils to each
lab session.
Lab reports:
Your lab reports will be written on 8.5 x 11” lined paper. Use
quad paper and a ruler for all graphs and tables. All labs must
be written in pen, and presented in the order instructed. You
do not need to restate the question. You may bullet point your
answers instead of using complete sentences, unless you are
writing in paragraph format. Typed lab reports are acceptable
as well.
General lab
instructions:
Every lab has unique instructions for the lab report, which can
be found under the “General Instructions” heading. Make sure
you follow these instructions, and write up your lab in order.
Make sure you include data from each section of the lab (worth
2 points per section!). The labs will be performed in groups,
but your reports must be individual and unique work. Any
shared or copied entries will receive a zero grade.
Reviewing
your lab:
At the end of each lab period, after your lab work is complete
and handed in, you may view the answer key. The answers are
my property; do not copy or photograph them, and do not
share them with other students. Copying the answers, adjusting
your lab report, or sharing answers with other students will be
considered a violation of the Student Honor Code, and will be
handled accordingly.
Grading:
Your labs will be graded regularly during the semester. Any
work that is not affixed to your lab will not be graded.
Physiology 3 Lab Manual, Spring 2016
10
Formal Lab Reports
In this course, you will be instructed to write 2 formal lab reports: one for the
nervous system lab, and one for the urinary system. Follow this format for each
formal lab report:
1. Name and Date: (and group members for the ANS lab)
2. Title: Make sure that the title is descriptive.
3. Introduction: A detailed 1-3 paragraph explanation of the physiology being
explored in the lab. Include your hypothesis for the experiment and the
related mechanism. Use complete sentences.
4. Procedure: Briefly list the steps that were actually taken. List statements in
past tense. Do not use personal pronouns.
5. Data: This section contains the raw data results of the experiment as well
as statistics (mean, standard deviation, p-value). It should also include a
graph of means with standard deviation bars. The graph must be labeled,
and must be made with a ruler or a computer.
6. Discussion: A detailed 1-3 paragraph explaining the results. What do your
observations tell you? What do your statistics indicate? How does this fit
into the context of the physiological concept being explored? What is
probable mechanism behind your findings? How robust are the results, given
their statistical and biological significance? What are potential sources of
error and how could they have affected the data?
7. Questions: Answer any questions posed in the lab manual.
Physiology 3 Lab Manual, Spring 2016
11
Lab 1: Introductory Lab
Goals: To become comfortable with:
1. Scientific notation, scale, and significant figures
2. Solute concentrations
3. The scientific method
4. Using a pipetman
5. Spectrophotometer use
6. Graphing and the standard curve
Background:
The concepts listed above are fundamental to the study of physiology, and will
be used in experiments in this class. Familiarity with these concepts is crucial
to allowing you to complete and understand the labs in this course.
General Instructions:
No pre-lab, this week only. However, it is wise to work through as much of this
lab as possible beforehand. Answer the questions in order.
Activity 1: Understanding scientific notation, scale, and significant figures
1. Scientific notation
The numbers used in physiology are typically very large or very small. For
example, the amounts of substances found inside cells are very small yet
the numbers of molecules are still quite large. The most convenient way to
express this great range of values is by scientific notation, in which numbers
are expressed as products of a number times ten to some power. Values less
than one and greater than zero are expressed with negative exponents,
while values greater than one are expressed with positive exponents:
0.1 = 10-1
0.01 = 10-2
0.001 = 10-3
1 = 100
10 = 101
100 = 102
1000 = 103
For example: the number 367 can be expressed as 3.67 x 102. The number
0.052 can be expressed as 5.2 x 10-2.
12
Physiology 3 Lab Manual, Spring 2016
2. Scale
10-9 L
10-6 L
10-3 L
1L
103 L
nL
ųL
mL
L
kL
nanoliter
microliter
milliliter
liter
kiloliter
3. Precision, significant figures, and rounding off
Imagine that you have weighed out 88.8 g of NaCl, and that you would like
to calculate how many moles of NaCl that equals. You divide 88.8 g by
NaCl’s molecular weight of 58.44g/mol, and your calculator indicates that
the result is 1.5195071 moles. This impressive figure incorrectly suggests
that you were able to measure the number of moles to the nearest tenmillionth of a mole! In truth, your response cannot be more precise than the
initial reading. If your scale could only measure to a precision of three
significant figures, then all calculations derived from these measurements
can be no more precise than that. The significant figures of a number are
those digits that carry meaning contributing to its precision. This includes
all digits except leading and trailing zeros (all those before and after the
non-zero digits).
In the example above, the calculated figure will need to be rounded off to
three significant figures. The thousandths unit (1.5195071 moles) is no
longer a significant figure. If the thousandths unit were a 4 or lower, you
would round down (1.51 moles). Since the thousandths unit is a 6 or higher,
you would round up the hundredths digit to 2 (1.52 moles). If the
thousandths unit were a 5, you would alternately round up and then down.
Questions:
1. Convert standard notation to scientific notation: 0.0035
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 chloride? (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, Spring 2016
13
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, Spring 2016
14
Molality specifies both the solute and solvent amounts:
Molality (molal, or m) = moles of solute
kilograms of solvent
The solvent amount is specified in kilograms, because different solvents
have different densities. The solvent you will be using in this class is water,
which has a density of 1.0 kg/L. Therefore, if the solvent is water, the
above equation can be simplified: molality = moles solute /L water.
Later in the course you will also come across the term “osmolality (Osm).”
This is used to describe the molality of a solution that has a several
different types of solutes, as in a body fluid. Osmolality adds the moles of
the various solutes and expresses that as a ratio to the amount of solvent.
Molality example:
a. If you measure out 1.0 mole of sucrose (342.3 g) and mix it with exactly
1.0 L of water, what is the resulting molality?
Density of water = 1.0 kg/L, so 1.0 L weighs 1.0 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 volume
of solution. A 1% solution is defined as 1 g solute per 100 mL solution.
Percent solution example:
a. A 4% NaCl solution is made by adding 4 g of NaCl to 100 mL solution.
Questions: Show your calculations. Units (M, m, g, L) are required throughout
the course.
5. 80.0 g glucose (molecular weight 180 g/mol) is dissolved in enough water
to make 1.0 L solution. What is the molarity of the solution?
6. You are planning on making 1.0 L of a 0.5 M solution of sucrose
(molecular weight = 342 g/mol). How much sucrose do you weigh out?
7. You are planning on making 1.0 L of a 0.5 M solution of glucose
(molecular weight = 180 g/mol). How much glucose do you weigh out?
8. What is the resulting molality if 0.75 mol is dissolved in 2.5 L of water?
9. Which solution has more solutes, a 1.0 m solution of glucose or a 1.0 m
solution of NaCl? (Hint: NaCl ionizes)
10. If I ask you to make 1000ml of a 2% solution of glucose, how much would
you weigh out?
Physiology 3 Lab Manual, Spring 2016
15
Activity 3: Scientific method
The scientific method is a set of techniques that serves to answer scientific
questions. It involves the following systematic method of inquiry:
Hypothesis: A simple yet specific statement of the effect of the treatment.
HOW TO WRITE A HYPOTHESIS: “The [treatment] will [have a specific
effect] on [parameter of interest] compared with the [control]. For
example: Drinking 16 oz. of Gatorade decreases urine volume compared
with drinking the same amount of water.
Experiment: The hypothesis is tested by designing and performing an
experiment, which often involves testing effects of a treatment. A welldesigned 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. Ex: urine volume
Conclusion: Either validation or invalidation of the hypothesis based on the
results of the experiment. Analysis of the effect of a treatment centers around
statistical significance (are the groups statistically different?), biological
significance (is the difference biologically relevant?), and scope of inference
(was the experiment only performed on middle-aged Caucasian women?).
Repeated verification of a hypothesis may result in a theory. Eventually, the
theory may become a law, or principle.
Questions: Consider the following experiment (reference below). It has long
been thought that antioxidants decrease risk for developing cancer. This was
tested by giving either the antioxidant Vitamin E or placebo at random to
35,000 men, and measuring how many were diagnosed with prostate cancer 5
years later. There was no statistically significant difference in prostate cancer
rate between the groups at the end of the study.
11. What was their hypothesis? (Be specific)
12. What was their treatment group?
13. What was their control group?
14. What was the dependent variable?
15. Can they broadly conclude that ALL antioxidants do not prevent ALL
cancers? Why or why not? **Careful, this is tricky.
Reference: Lippman SM et al. Effect of selenium and vitamin E on risk of
prostate cancer and other cancers. JAMA. December 9, 2008.
16
Physiology 3 Lab Manual, Spring 2016
Activity 4: Working with a Pipetman
You will frequently be using a pipetman in lab to measure exact volumes of
solutions. We have four sizes of pipetman in this lab, each with a range of
volumes that it can measure. Acquaint yourself with these volume ranges:
Pipetman
P20
P200
P1000
P5000
Maximum volume
.02 mL (20 ųL)
.2 mL (200 ųL)
1 mL (1000 ųL)
5 mL (5000 ųL)
Range
1 – 20 ųL
20 – 200 ųL
200 - 1000 ųL
1 - 5 mL
There is a dial on each pipetman, which you will turn to set the volume in
microliters. Each volume display has three numbers in it. If you were to dial
the pipetman to maximum volume (for example, to 200ul in the P200), the
topmost number in the display corresponds with the first digit (2), the second
with the second digit (0), and the third with the third digit (0). Familiarize
yourself with the display and volumes of each of the four types of pipetman:
Pipetman Instructions:
1. To adjust the volume, turn the volume adjustment knob until the digits
represent the volume that you need to pull up. The top number should
not exceed the first number of the pipetman (2 for P20 and P200; 1 for
P1000, and 5 for P5000). For example, 5 mL looks like “500” in a P5000,
1000 ųL looks like “100” in a P1000, and 50 ųL looks like “050” in a P200.
2. Attach a disposable tip by pressing the pipetman firmly into the base of
the tip without touching either the pipetman or the tip. Use the correct
Physiology 3 Lab Manual, Spring 2016
3.
4.
5.
6.
17
tip size (the tip color should match the color on the top of the
pipetman).
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.
Avoid getting fluid into the pipetman: keep your pipetman vertical at all
times, and pull fluid up slowly.
The same tip can be used repeatedly in the same reagent, from low to
high concentration. Be careful not to contaminate.
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?
18. If you take the P20 and dial “020” from top down, how much fluid are
you pulling up?
Activity 5: 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.
Physiology 3 Lab Manual, Spring 2016
18
In Activity 7, you will be making a
standard curve (actually, a line), which
is a particular type of line graph that
relates two measures, in this case
absorbance units from a spectrophotometer and solute concentration.
Absorbance values of known
concentrations of product are recorded
and graphed. A straight line is drawn
that best incorporates all of the points
on the graph and goes through the
origin. The points themselves need not
fall on the line. In a future experiment,
when unknown concentrations of solute are placed in the spectrophotometer,
the standard curve is used to relate absorbance readings to solute
concentration.
Activity 6: Spectrophotometer Use
A spectrophotometer 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)).
We will be using a spectrophotometer to measure the concentration of product
produced in the enzyme lab. Before we can do that however, we must first
calibrate the instrument. This is done in order to correct for any background
absorption by the solvent medium as well as the cuvette material. Refer to the
following instructions when instructed to calibrate the spectrophotometer.
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.
4. Using the knob on the left (the same one you used to turn on the
spectrophotometer) adjust the needle or number to zero transmittance.
5. 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.
6. The spectrophotometer is now calibrated.
19
Physiology 3 Lab Manual, Spring 2016
Activity 7: Making a P-nitroaniline Standard Curve
In this activity you will make a standard curve for p-nitroaniline in groups of 35 students. P-nitroaniline is a product made by the enzyme trypsin. This
standard curve will be used in Lab 2.
1. Obtain 5 cuvettes and label them 1-4 and B with a wax pencil.
2. Pipette the following into the indicated tube:
Tube
1
2
3
4
B (blank)
10-4 M pnitroaniline
2.50 mL
0.50 mL
0.25 mL
0.05 mL
0.00 mL
Tris buffer
solution
2.50 mL
4.50 mL
4.75 mL
4.95 mL
5.00 mL
Final molar concentration
of p-nitroaniline
5.0 x 10-5
1.0 x 10-5
0.5 x 10-5
0.1 x 10-5
0
3. Calibrate the Spectronic 20 (see Activity 6).
4. Mix your solutions with the micro vortex. Visually confirm that all of the
cuvettes have equal volume of solution.
5. Read the absorbance of tubes 1-4. Check that the data trend makes
sense before you continue. No values should be zero.
6. Record your results in a table in your lab notebook. Make sure that your
table has the concentration information from the table above. (see
example at the end of Lab 2) (2 points)
7. Plot a standard curve of absorbance v. p-nitroaniline concentration (see
example in Activity 5; p-nitroaniline is “X”). Draw a straight line that
best fits the points and goes through the origin. The line does not have
to actually go through any of the points, but it must be an average of all
of the information. Make a copy or photo of this graph because you will
need it for the pre-lab next week. (5 points)
Physiology 3 Lab Manual, Spring 2016
20
Lab 2: Enzyme Lab
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-pnitroanilide HCl (BApNA). BApNA will be hydrolyzed by trypsin. When BApNA is
hydrolyzed, a yellow substance called p-nitroaniline will be released, and this
can be measured colorimetrically, using a spectrophotometer. Today’s
experiment involves measuring the amount of BApNA that is being catalyzed by
trypsin, and testing which factors affect the activity of trypsin.
General Instructions:
You will carry out these experiments in groups of four. Each group will be
assigned one of the experiments to do. Record all data and make a graph for
the experiment you conduct. Pay attention to units throughout. Use graph
paper and a ruler for tables and graphs. Answer the questions neatly and in
order at the end of your lab.
Pre-lab: (10 points)
 In your own words, define the following: enzyme, substrate, product, pH,
absorbance, and transmittance

Answer the following questions:
1. Which substance are you measuring with the spectrophotometer?
2. What will the slope of the standard curve allow you to calculate?
3. Attach an image of your standard curve from Lab 1. Calculate the slope
of your standard curve. Show your calculations and include units. An
example can be found at the end of this lab. Make a note, copy or photo
of this slope; you will need it later in this lab.
4. Use the slope of your line to calculate the concentration of solute if the
absorbance is 0.15 AU. Show your calculations.
21
Physiology 3 Lab Manual, Spring 2016
Experiment 1: Effect of substrate concentration on reaction rate
1. Obtain 6 cuvettes and label them 1-4 and B.
2. Pipette the following into the indicated tubes:
Tube
10-3 M BApNA
1
2
3
4
B (blank)
5 mL
4 mL
3 mL
2 mL
2.5 mL
Tris buffer
solution
0 mL
1 mL
2 mL
3 mL
2.5 mL
Final molar
concentration of BApNA
1.0 x 10-3
0.8 x 10-3
0.6 x 10-3
0.4 x 10-3
0.5 x 10-3
3. Visually confirm that all tubes have the same volume of solution.
4. Place the tubes for 2 minutes in the 37ºC incubator.
5. To tube B add 0.1 mL of 0.001 M HCl. Mix and wipe the cuvette with a
kimwipe. Calibrate the spectrophotometer using tube B as a blank.
6. Add 0.1 mL trypsin enzyme to tubes 1-5 and mix by shaking the tubes.
7. Record the time. Return the cuvettes to the incubator and record
absorbance values every 3 minutes for a total of 9 minutes in a table
format. Make sure to dry and mix the cuvettes each time before
measuring absorbance. Also make sure that absorbance readings go down
as tube number goes up, that all readings are non-zero, and that all the
absorbance readings uniformly go up with time.
8. Convert each absorbance to concentration using the slope of the
standard curve that you calculated in your pre-lab (see example at end
of lab). Record the concentrations in a table.
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. Place 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
1
2
3
4
B (blank)
BApNA
5.0 mL
5.0 mL
5.0 mL
5.0 mL
5.0 mL
Temperature
Ice (0-5ºC)
Room temperature (22-25ºC)
Body temperature (37ºC)
Boiling water (100ºC)
Room temperature (22-25ºC)
22
Physiology 3 Lab Manual, Spring 2016
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. Boil 0.2 mL trypsin and then add 0.1mL of it to tube 4. The goal of this
step is to add boiled trypsin to tube 4.
5. Add 0.1 mL room temperature trypsin enzyme solution to tubes 1-3 at
the same time as step 4. Record the time.
6. Immediately return the cuvettes to their respective temperatures. Wait
for 3 minutes. Then remove a cuvette, mix it, and wipe dry with a
kimwipe. Read the absorbance of the tube and immediately return the
cuvette to its incubator. Repeat for the rest of the tubes.
7. Measure absorbance of all tubes every 3 minutes for a total of 9 minutes
and record these data in a table. Make sure that you mix and dry each
tube before measuring. Make sure that all absorbance readings are nonzero, and go up with time.
8. Convert absorbance to concentration using the slope of the standard
curve calculated in the pre-lab (see end of lab for example calculations).
Record the concentrations in a table.
9. Plot end-product concentration v. time for all four curves on one graph.
Distinguish the lines using different colors. Connect the points.
Experiment 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
1
2
3
4
5
B (blank)
BApNA
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
pH buffer solution
4 mL pH 6
4 mL pH 7
4 mL pH 8
4 mL pH 9
4 mL pH 10
4.0 mL Tris
3. Wait for 2 minutes
4. 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.
5. Add 0.1 mL trypsin solution to tubes 1-5. Record the time and place each
in the incubator at 37 ºC.
6. At 5 and 10 minutes, read the absorbances of the tubes and record the
results in a table. Make sure to mix and wipe the tubes dry with a
kimwipe before each spectrophotometer reading. Make sure that all
absorbance readings are non-zero, and that they go up with time.
7. Convert each absorbance to concentration using the slope of the
standard curve calculated in the pre-lab (see example at the end of lab).
Record these concentrations in a table.
Physiology 3 Lab Manual, Spring 2016
23
8. Plot end-product concentration v. pH. Plot two separate lines, one for 5
minute and one for 10 minute readings. Distinguish the lines using
different colors or dashed lines. Connect the points.
Questions:
1. Why did increasing the substrate concentration increase reaction rate?
2. Why did reaction rate increase with temperature up until body
temperature?
3. Why did reaction rate decrease towards boiling?
4. Why was the pH optimum basic? Why does the enzyme not function well
at pHs that are lower than the optimum? (don’t restate the question)
5. Pepsin is a stomach enzyme that breaks down proteins into amino acids.
List its optimum temperature, pH, and protein concentration.
6. Describe an experiment that you could do to test another factor
affecting this enzyme.
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.
24
Physiology 3 Lab Manual, Spring 2016
Example Enzyme Lab Tables, Graphs, and Calculations:
Activity 1: Standard Curve
Example data:
Tube
1
2
3
4
B (blank)
Final molar
concentration of pnitroaniline
5.0 x 10-5
1.0 x 10-5
0.5 x 10-5
0.1 x 10-5
0
Absorbance Units
(AU)
.48
.11
.05
.01
0
Standard Curve
Slope of this line*: __.1AU_______ = .1 AU/Mx10-5
1 x 10-5 M
* The exact number of your slope will depend on your standard curve. It is
easiest to keep Mx10-5 as part of your units, so that you can just divide each AU
data point by .1, and all resulting concentrations will have the units Mx10 -5.
25
Physiology 3 Lab Manual, Spring 2016
Experiment 1, 2, or 3
Example data:
Tube
1
2
3
4
AU at 2 minutes
.2
.25
.3
.35
AU at 4 minutes
.3
.35
.4
.45
Convert each AU into concentration Mx10-5 (divide each AU by your slope):
Tube
1
2
3
4
Mx10-5 at 2 min
2.0
2.5
3.0
3.5
Mx10-5 at 4 min
3.0
3.5
4.0
4.5
Now graph the concentration of your product (put units on the axes):
Physiology 3 Lab Manual, Spring 2016
26
Lab 3: Osmosis Lab
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.
General Instructions:
You will perform experiments in groups of 3-4. Record all data and draw a
graph for Activity 2. Answer the questions in order at the end of the lab.
Pre-lab: (8 points)
 Define the following: osmosis, osmotic pressure, hypertonic / hypotonic,
hydrophilic / hydrophobic

Using the information about osmolality below, answer the following
questions:
1. Which of the following solutions has the greatest osmotic pressure (i.e.
greatest number of moles): 30% sucrose (MW = 342g/mol), 60% sucrose,
or 30% magnesium sulfate (MW = 246.4g/mol)? Why? Show your
calculations. (Magnesium sulfate is ionic. The definition of a percent
solution can be found in Lab 1.) (2 points)
2. Does 30% NaCl (MW = 58.44g/mol, ionic) have the same, increased, or
decreased osmotic pressure as 30% magnesium sulfate? Why?
3. Saline infusions are often provided medically to rehydrate individuals. It
is only meant to increase blood volume, not to change the ratio of solute
to solvent. What is the osmolality of the solution?
Osmolality
Osmolality is a measure of the number of solutes in a given amount of solvent.
Osmolality (Osm) = osmoles of solute
kilograms of solvent
If 180 g of glucose and 180 g of fructose are dissolved in the same liter of water
(both have molecular weight of 180 g/mol), the osmotic pressure, or the
pressure driving water into a solution, would equal that of a 360 g/L glucose
Physiology 3 Lab Manual, Spring 2016
27
solution. Osmolality does not depend on the chemical nature of the solutes,
but rather on the number of solutes. The molality of this sugar solution is 2.0
m, but is written as 2.0 Osm, because it contains more than one type of solute.
When electrolytes like NaCl ionize in water, the number of solutes, and thus
the osmolality, is higher than that of a nonionizing solute (double, for NaCl).
Blood plasma osmolality is equal to 0.3 Osm, or 300 mOsm. Solutions that have
the same osmolality as plasma are said to be isosmotic (equal osmolality), or
isotonic (equal pressure) to plasma. Solutions that have a higher concentration
of solute than that of plasma are called hypertonic, and those that have a
lower concentration of solute are called hypotonic.
Activity 1: Effect of solute concentration on cell membranes
1. Obtain 5 test tubes. Label them. Put on gloves.
2. Add 2 mL of solution 1 to tube 1, and record the solute content of the
solution. Place 2 mL of solution 2 into tube 2, and so on for all 5 tubes.
3. Add one drop of sheep blood to each test tube. Mix well by shaking.
4. Using a transfer pipette, place a drop of solution 3 onto a slide, and
cover it with a coverslip. Observe the cells at 400x on the microscope.
Do the same for the other solutions and draw your results. Make sure to
cover your work area with a paper towel.
Questions:
1. Which solution was likely isotonic?
2. Based on your observations, would a cell in a .2% saline solution be in a
hyper-, hypo-, or isotonic solution?
3. What would happen to the cell in question #2, and why?
4. Calculate the percent saline (NaCl) solution that would be isotonic. The
molecular weight of NaCl is 58.44 g/mol. Show your calculations.
(Remember that NaCl is ionic.) (2 points)
Activity 2: Osmosis across an artificial semipermeable membrane
1. Three solutions will be prepared for you and placed in an artificial
semipermeable membrane (“dialysis tubing”): 30% sucrose, 60% sucrose,
and 30% magnesium sulfate (MgSO4).
2. The dialysis tubing will be placed in a beaker of water.
3. Record the height of the column of water above the second string in cm
every 10 minutes for 60 minutes.
4. Draw one graph of column height over time, representing the different
solutions with different colors.
Physiology 3 Lab Manual, Spring 2016
28
Questions:
5. Why did the columns rise? Describe the mechanism.
6. Using your graph, calculate the osmosis rate for each solution (cm/time)
at the start.
Example:
Initial rate: 60.0cm = 2.0 cm
30.0 mi
min
7. Which column rose the most?
Why? Was that expected?
Why, or why not?
8. The osmosis rate in these set-ups will slow down over time. Why? (They
will not become isosmotic because solutes will never leave the bag)
9. Is sucrose hydrophilic or hydrophobic? Polar or nonpolar?
Disposal
Used slides should be placed in the disinfectant bucket. Test tube contents can
be dumped into the labeled waste container, and tubes placed in the glass
disposal bin. Place used transfer pipettes and soiled gloves in the biohazard
container. Clean your work area with disinfectant.
Physiology 3 Lab Manual, Spring 2016
29
Lab 4: Group Presentation
There is no pre-lab for this. 8% of your grade is based on a group project.
You will work in groups of 3 to explore a new scientific finding and present
the finding and its underlying physiology to the class. All groups will present
their work during lab at the end of the semester. Today you will be assigned
your group and topic, and we will go over statistics for physiology.
Your group will be assigned a specific system on which your presentation
must be focused. Find 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 *only what is needed to
understand your presentation
- Analysis of the original research article
o Introduction to the question investigated
o Methods
o Results (include data figures here, point out important numbers)
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 me a pdf of the journal article and a paper copy before the 8th
week of class. You are welcome to first email the citation or abstract to
make sure that it is an appropriate article.
You will be graded as follows: background physiology (10), methods (10),
results (10), conclusions (10), critique (10), quality of presentation (10),
encouraging student involvement (10), and participating in other
presentations (5), for a total of 75 points.
Good luck, and have fun with this!
Physiology 3 Lab Manual, Spring 2016
30
Lab 5: Neurobiology Lab
Goals:
1. To become familiar with reflex circuitry in the nervous system
2. To understand the effect of sensory stimuli on efferent output
3. To become familiar with designing, executing, and communicating a
scientific research project
Background:
The autonomic nervous system (ANS) is part of the peripheral nervous system.
Its goal is to maintain homeostasis of the visceral organs. In this lab we will
examine the mammalian diving reflex, which involves the ANS. In mammals,
submerging the face in water initiates several physiological responses that
maximize the time that can be spent under water. This includes a reduction of
heart rate (bradycardia). The reflex arc is as follows: the trigeminal nerve
(CN5) of your face, nose, and mouth senses the cold temperature of the water,
relays this information to the brainstem, which then sends parasympathetic
efferent instructions to the heart via vagus nerve (CN10). In humans, mild
bradycardia is also caused by breath-holding without submersion. The
physiological goals of the diving reflex are to reduce the energetically costly
aerobic activity of the heart.
General Instructions:
Work in groups of 4-6. Design an experiment to test the diving reflex. At your
disposal you have pulse oxymeters (to measure heart rate), snorkel masks,
buckets, and different temperatures of water. All individuals in your group will
dive twice, one for the control and once for the treatment (alternate the order
and rest in between dives). Dive lengths are 40 seconds. Please limit your
stimulant use in the hour prior to class (eg. caffeine). Write up one formal lab
report for your group. Follow instructions for writing a formal lab report found
near the start of this manual. You are welcome to analyze the statistics
together with me on the class computer.
Pre-lab: (5 points)
 Define the following terms: autonomic nervous system, bradycardia, reflex
arc

Answer the following questions:
1. Describe the two nerves involved in the reflex arc that you are testing
today.
2. Describe the neurotransmitters and receptors used by this two-neuron
efferent pathway (both at the ganglion and at the target).
Physiology 3 Lab Manual, Spring 2016
31
Questions:
1. Why was replication (having a sample size larger than 1) beneficial to
your experiment?
2. If one of your classmates drank caffeine before taking part in the
experiment, it would introduce bias. Which kind of bias would it
introduce, and why is that problematic for data analysis? (We covered
this in statistics; see summary below)
3. If every member of your group performed the treatment condition first
and the control condition second, it would introduce bias. Which kind of
bias would it introduce, and how does that affect data analysis?
Bias: There are two main types of bias. Random bias is due to sampling
variability and is quite normal (eg. you are testing the effect of exercise
on heart rate, but subjects have varying levels of fitness to begin with).
This is can be addressed by statistical analysis and is usually not a
serious concern. It manifests as an increase in variability, which makes it
more difficult to see differences between groups. Systematic bias is far
more lethal to a study; this is caused by a factor that affects one group
differently than the other (eg. you are testing the effect of exercise on
heart rate and place elderly in the exercise group and teenagers in the
non-exercise group). With this type of bias, you are no longer able to
determine the effect of the treatment, because the effects are
confounded by another variable.
Physiology 3 Lab Manual, Spring 2016
32
Lab 6: Sensory Physiology Lab
Goals:
To become comfortable with the mechanisms underlying sensory physiology and
the ways in which some of these sensations are tested.
Background:
The senses include cutaneous, gustatory, olfaction, vestibular, auditory, and
visual senses. Specialized receptors, such as chemoreceptors, mechanoreceptors, thermoreceptors, and photoreceptors, sense information about the
external world and send neural impulses to the brain. This allows us to
interpret the world around us. In today’s lab, you will explore your own sensory
physiology and explore the mechanisms underlying each sense.
General Instructions:
Work in teams of 2, and perform each of these tests on yourselves. Record your
own data in your lab and answer questions in order at the end.
Pre-lab: (9 points)
 Define the following terms: sensory receptor, receptive field, retina,
cochlea, semicircular canal

Answer the following:
1. Describe why two cutaneous points can be sometimes felt as two, and
sometimes as one.
2. Does light striking a photoreceptor result in its depolarization or
hyperpolarization? Why?
3. Describe how a hair cell is bent upon hearing sound.
4. Describe how the vestibular system is stimulated by spinning.
Physiology 3 Lab Manual, Spring 2016
33
Activity 1: Cutaneous Senses
1. Two-point discrimination
Use the calipers to determine the two-point threshold of your
partner’s palm, back of the hand, fingertip, and back of the neck.
Start with the calipers wide apart and the subject’s eyes closed.
Randomly alternate two-point with one-point contact so that your
partner can’t anticipate you. The partner tells you whether they feel
one or two contacts. Decrease the distance between the needles
until you partner can no longer accurately tell you how many points
are touching. You partner records this distance, and you proceed to
another body part. When you are done, switch roles.
Question:
1. Which body part had the smallest receptive field, and how do you
think that correlates with the size of its neural representation in
the homunculus?
2. Referred pain
Use the reflex hammer and gently tap the ulnar nerve where it
crosses the medial epicondyle of the elbow. Describe the location
where you perceive tingling or pain.
Question:
2. Describe the two mechanisms for referred pain discussed in class.
Which of the two is at work in this example?
Activity 2: Vision
1. Visual acuity: Snellen eye chart
Stand 20 ft. away from the Snellen eye chart. Covering one eye,
attempt to read the line with the smallest letters you can see (with
glasses off, if you wear contact, you can leave them on). Your
partner can determine your visual acuity. Record this in your
notebook. Repeat this procedure with the other eye.
Question:
3. If you looked at the 20/20 line and it was in focus, where in the
eye was the image projected onto? If a person’s eyesight is worse
than 20/20, where is that image projected?
Physiology 3 Lab Manual, Spring 2016
34
2. Color vision
Use the color-blind tests provided, which are a series of colored dots
arranged in circles. Look at these. A person with normal vision will
see a number embedded within each circle.
Question:
4. Why would a color-blind individual see something different?
3. Blind spot
Follow the instructions on the blind-spot card.
Question:
5. What is the physiological basis for the blind spot? (Be specific)
4. Bleaching
For 1 minute, stare at the dot on the red paper, keeping your head
steady. Then suddenly shift your gaze to a sheet of white paper.
Repeat this for the blue square.
Question:
6. What is the physiological explanation for this illusion? Be specific.
Hint: white light contains all of the wavelengths of visible light.
Activity 3: Hearing
1. Weber’s test
Place the handle of the vibrating tuning fork on the midsagittal line
of your head and listen. In conduction deafness, the sound will seem
louder in your affected ear because it is not competing with outside
noises, but in sensory deafness, the sound will be louder in the
normal ear. Repeat this test with one ear plugged. On which side is
the tuning fork louder?
Question:
7. Explain why the sound is louder in the affected ear in conduction
deafness, and louder in the normal ear in sensory deafness. Use
what you know about the underlying causes of these two
pathologies.
Physiology 3 Lab Manual, Spring 2016
35
Activity 4: Vestibular System
1. Vestibular-ocular reflex
Have the subject sit in a swivel chair with the eyes open and head
flexed forward (chin almost touching chest). Rotate the chair quickly
to the right for 20 seconds. (Do not do this if you get sick) Abruptly
stop the chair and have the subject open eyes as wide as possible.
Note the direction of nystagmus. (Subject should then close eyes
until dizziness goes away).
Question:
8. How does spinning result in nystagmus?
Physiology 3 Lab Manual, Spring 2016
36
Lab 7: Scientific Article
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
General Instructions:
We will be analyzing a scientific research journal article (both the PowerPoint
and pdf will be posted). Please read the article before coming to class, and
print and bring the article to class. At the end of the discussion, you will write
a critique of the paper.
Pre-lab: (5 points)
 Read the assigned article

Write a one paragraph summary of the study

Prepare a rough draft list of strengths and weaknesses of this article (this
will be turned in with the pre-lab so make a copy if you need it)
Paper Critique Instructions
Write an essay in which you critically analyze this research article. Consider
biological significance, scope of inference, study design, and whether the
conclusions drawn are valid. Make sure that your review is organized,
thoughtful, and neat. Your essay should include the following sections:
1. A paragraph summarizing the study (done in the pre-lab). (5 points)
2. A paragraph discussing its major strengths. (5 points)
3. A paragraph discussing its major weaknesses. For each weakness,
support your comments. Describe why it is a weakness and what the
author should have done differently. This is the meat of your essay, so
give it some thought. (10 points)
4. A final paragraph giving your overall opinion of this article as a scientific
work. (5 points)
Physiology 3 Lab Manual, Spring 2016
37
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.
General Instructions:
Perform these experiments in groups of 3-4. Perform all 3 experiments in
parallel to save time. Record your data and answer questions in order at the
end.
Pre-lab: (10 points)
 Define the following: digestion, hydrolysis, absorption, secretion, bile, bile
salt, emulsification

Answer the following questions:
1. What are the actions and optimal pH and temperature for salivary
amylase?
2. What are the actions and optimal pH and temperature for pepsin?
3. What are the actions and optimal pH and temperature for pancreatic
lipase?
Physiology 3 Lab Manual, Spring 2016
38
Experiment 1: Digestion of Carbohydrates
1.
2.
3.
4.
5.
6.
Label 4 clean test tubes 1-4.
Obtain 9 mL of saliva in a small, graduated cylinder.
Add 3.0 mL distilled water to tube 1.
Add 3.0 mL saliva to tubes 2 and 3.
Add 3 drops of HCl to tube 3.
Boil the remaining saliva in a separate Pyrex test tube for 5 minutes. Let
it cool. When cool, add 3.0 mL of this boiled saliva to tube 4.
7. Add 5.0 mL cooked starch to each of the test tubes.
8. Mix these tubes well by shaking or vortexing.
9. Incubate all tubes for 1 hour at 37ºC.
10. Mix the tubes well again.
11. Divide each sample by pouring half of each into four new test tubes.
12. Test one of the sets of four solutions for starch by adding a few drops of
iodine to each tube. A purplish black color indicates presence of starch.
13. Test the other four solutions for monosaccharides and disaccharides by:
a. Adding 5.0 mL Benedict’s reagent to each of the four test tubes
and immerse them rapidly in boiling water bath for 2 minutes.
b. Remove the tubes from the boiling water and rate the amount of
simple sugars according to the following scale:
i. Blue (no monosaccharides and disaccharides)
ii. Green (very little)
iii. Yellow (some)
iv. Orange (significant)
v. Red (a lot of monosaccharides and disaccharides)
Questions:
1. What were the effects of adding HCl on amylase activity? Why?
2. What was the effect of boiling on the activity of amylase? Why is that?
3. Which of the experimental conditions do you think most closely mirrors
what happens in your mouth? Why?
Experiment 2: Digestion of Protein
1. Label 4 clean test tubes 1-4. Cut 4 slices of egg white about the size of a
fingernail and as thin as possible. They must be uniform in size and ultra
thin. Place one slice in each of the 4 test tubes.
2. Add 1 drop of distilled water to tube 1.
3. Add 1 drop of HCl to tubes 2, 3, and 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.
Physiology 3 Lab Manual, Spring 2016
39
7. Add 5.0 mL chilled pepsin to tube 3 (place pepsin in ice first), and place
tube 3 in an ice bath as soon as possible.
8. Leave all tubes for 1 hour, remove the tubes.
9. Record the appearance of the egg white.
Questions:
4. Under which pH conditions did pepsin work best? Why?
5. Did pepsin act more successfully in ice or at 37ºC? Why?
Experiment 3: Digestion of Triglycerides
1. Label 4 clean test tubes 1-4. Add 3.0 mL litmus cream to each tube.
Cream is rich in neutral triglycerides. Litmus is a pH indicator which is
blue in alkaline conditions and red in acidic conditions.
2. Add 6.0 mL distilled water to tube 1.
3. Add 3.0 mL bile extract and 3.0 mL distilled water to tube 2.
4. Add 3.0 mL pancreatic lipase and 3.0 mL distilled water to tube 3.
5. Add 3.0 mL pancreatic lipase and 3.0 mL bile extract to tube 4.
6. Mix the tubes.
7. Incubate these at 37ºC for 1 hour. Shake and record any color changes.
Questions:
6. Based on your results, which secretions are needed to digest
triglycerides?
7. Describe the role that bile extract has in digestion.
8. In the triglyceride experiment, why does the post-digestion pH decrease?
(FYI: bile is neutral and pancreatic juice is basic)
9. What do you think the resulting color would have been in tube 4 if you
had performed the experiment on ice? Why?
Disposal
Benedict’s and litmus solutions should be disposed of in the waste container.
All other solutions can be disposed of down the drain. Place all pipetman tips in
the trash, and all glassware in the soaking bucket in the sink.
Physiology 3 Lab Manual, Spring 2016
40
Lab 9: Blood Lab
Goals:
1. To become familiar with the various types of cell found in blood
2. To understand the physiology underlying blood typing
Background:
Centrifuging blood results in a separation of discrete layers. The top layer
consists of plasma, and the bottom layer contains formed elements. We will be
performing both a hematocrit and a cell count. We will also practice blood
typing, which is an important test for compatibility of blood types, and is based
on antigens on the surface of red blood cells.
General Instructions:
Perform these experiments in groups of 3-4. No open-toed shoes permitted.
Record all data and answer questions in order at the end.
Pre-lab: (10 points)
 Define: erythrocyte, leukocyte, thrombocyte, plasma, antigen, antibody,
agglutination

Answer the following questions:
1. What are the three categories of formed elements and for each, briefly
what is its function?
2. What does the hematocrit measure, and why is it important?
3. How would the mixing of incompatible blood types cause health
problems?
Physiology 3 Lab Manual, Spring 2016
41
Activity 1: Blood cell count
Obtain a slide of blood cells and a microscope. Look at blood at 400x
magnification and identify all of the three major formed elements in one field
of view. Count the number of each type of element in a field of view using a
counter. Specifically name and count the leukocytes that you see. You are
welcome to count all cells in a quadrant of the field and multiply by 4.
Questions:
1. Which formed element is most numerous?
2. Which kinds of leukocytes did you see, and what are their roles?
3. What does it mean when leukocyte count is high?
4. What is smallest formed element?
Activity 2: Hematocrit
Put on a pair of gloves. Obtain a capillary tube and place its tip in the sheep
blood, allowing the blood to move up the tube by capillary action. Then cap
the bottom (blood side) with sealing clay. Place the capillary tube in the
centrifuge, clay side facing out, along with at least one from another group so
that they are balanced. Close the lid and start the centrifuge at the MHCT
setting. When it is done, measure the hematocrit with the ruler provided and
record this number in your data section.
Questions:
5. Normal sheep hematocrit is 30-35%. State one health-related
circumstance that could cause the sheep hematocrit to be too low.
6. State one experimenter error that could cause the measured sheep
hematocrit to be too low.
Activity 3: Blood typing
You will be using fake blood to ascertain the blood type of 4 fake individuals:
1. Obtain a blood test card. Place a drop of anti-A (A antibodies) in the
circle with that label. Replace the cap (always do this if you are not
using the bottle).
2. Place a drop of anti-B (B antibodies) in its circle.
3. Place a drop of anti-Rh (Rh antibodies) in its circle.
4. Put a drop of blood cells in each circle.
Physiology 3 Lab Manual, Spring 2016
42
5. Rock the test card for 1 minute, but keep each mixture in its circle. Tilt
the card to drain the mixtures to the side of their circle, but not out of
their circles. Place the cards on a white paper. Look for agglutination
and record the blood type of each individual. The Rh reaction may take
as long as 5 minutes to take place.
6. In your data, show which combinations agglutinated.
Questions:
7. List all of the fake individuals with their blood types. (2 points)
8. Why did certain mixtures agglutinate? Use specific physiological terms
from lecture.
Disposal
Capillary tubes must be disposed of in the sharps container. Soiled gloves
must be placed in the biohazard container; those that are not soiled may be
thrown away. Fake blood test cards may be thrown in the garbage.
Physiology 3 Lab Manual, Spring 2016
43
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 challenged
3. To learn the origin of heart sounds and pulse
Background:
The electrocardiogram (ECG) measures waves of depolarization and
repolarization of the cardiac myocytes. As many as 12 different recordings can
be taken, each giving a different picture of the function of the heart. In this
lab, you will take a resting ECG of your heart using the bipolar limb leads. You
will analyze the wave segments of your ECG.
Blood pressure is the pressure that blood places on vessel walls, particularly,
the systemic arteries, during the systolic and diastolic activities of the heart. In
this lab, you will practice using the blood pressure cuff and stethoscope in
order to learn the underlying physiology, and then test which values change
upon lying down or exercising. Last, you will assess pulse rate and challenge it
to see if it changes.
General Instructions:
Work in groups of 2-4. Do each recording on yourself. Record your data from
each section and then answer the questions in order at the end.
Pre-lab: (10 points)
 Define the following: cardiac myocytes, pacemaker cells, cardiac systole/
diastole, blood pressure systole/ diastole

Answer the following questions:
1. Sketch out an ECG. Underneath that, sketch out the atrial and
ventricular myocardial action potentials, as they correspond with the
ECG. Underneath that, sketch out atrial and ventricular contraction, as
they correspond in time with the ECG. (Hint: Fig 13.21 and 13.22) (2
points)
2. Why is the QRS complex so much higher in amplitude than the other
waves?
3. Which interval represents the AV nodal delay? Why is that delay
important?
4. How do the Korotkoff sounds differ from lub/dub in terms of what
generates the sounds?
5. Why are high and low blood pressure dangerous?
Physiology 3 Lab Manual, Spring 2016
44
Activity 1: ECG
1. Preparing for the ECG:
Turn on the ECG. Clean both wrists and both ankles with an alcohol
swab. Attach an electrode sticker to each area, tab pointing down. Then
apply the following leads to these stickers: White – right wrist; Green –
right ankle; Black – left wrist; Red – left ankle. Make sure that the snap
tips are not touching the sticky part of the electrode.
Select the ‘(I, II, III)’ lead channel using the blue up and down arrows.
To start the ECG recording, press the ‘MANUAL PRINT’ button. Press the
red ‘STOP’ button to stop. You only need a few inches of recording.
Obtain your ECG strip. Paste the strip into your lab notebook under the
data section. Note that on the ECG, the x axis represents time (each
small box is 1 mm, or .04 s) and the y axis represents intensity (0.1 mV
per mm).
Questions:
1. On the Lead II section of your ECG strip, mark the following: (3 points)
a. P, Q, R, S, T waves
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.
2. Calculate your heart rate:
________1 beat __________ x __60 seconds__ = __# beats__
X seconds for one cycle
1 minute
minute
1 big box represents .2 sec, and each little box represents .04 sec.
Physiology 3 Lab Manual, Spring 2016
45
Activity 2: Blood pressure
1. Have someone record your blood pressure while you are sitting, lying
down, and after exercising. To take a manual blood pressure, a
sphygmomanometer and stethoscope are needed. Make sure to first
clean the stethoscope ear piece with an alcohol wipe (and wipe it down
after use as well). Wrap the cuff of the sphygmomanometer snugly
around the arm, above the elbow. Place the stethoscope diaphragm
under the cuff, over the brachial artery (just medial to the biceps
tendon). Inflate the cuff to about 180mm, using the bulb pump.
Inflation collapses the brachial artery, stopping blood flow. Deflate the
cuff gradually, at about 3mmHg per second. Watch the needle of the
display and listen for Korotkoff sounds, the sounds made by the blood as
it begins to move again. Systolic pressure is the pressure at which you
first hear sounds. Diastolic pressure is the pressure at which you first
hear no more sounds. Average blood pressure in a young healthy adult is
around 120/80. Make sure that you also become comfortable taking
other students’ blood pressure.
Questions:
3. Why are there no sounds with the stethoscope when the cuff is inflated
to 180 mmHg?
4. What is the basis for the Korotkoff sounds in the middle pressure range?
Describe in detail.
5. Why are there no more sounds when the cuff is deflated below 60
mmHg?
Activity 3: Pulse
1. Have a partner determine your heart rate by taking a pulse rate. Locate
the ulnar or radial artery at the wrist. Gently press against the vessel
with index and middle finger, adjusting slightly to locate the pulses.
Count the number of pulses that are felt in six seconds. Multiply that
number by ten to determine your heart rate (BPM = beats per minute).
2. Next, have your partner do something that will increase heart rate, and
measure again. You can exercise, ask an embarrassing or stressful
question, etc.
Questions:
6. What are you actually feeling when you measure your pulse?
7. Describe the mechanism responsible for the rapid rise in heart rate.
(neurotransmitters, receptors, channels)
Physiology 3 Lab Manual, Spring 2016
46
Lab 11: 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. The host cell will then express HIV
genes, resulting in the formation of new HIV particles that bud out of the cell.
During the early stages of infection, the HIV elicits humoral and cell-mediated
responses that result in circulating IgG molecules directed at specific viral
antigens. However, the virus has a high mutation rate, and many of the
variants survive and escape future immune detection.
Enzyme linked immunoabsorbent assay (ELISA) tests can detect specific
antibodies or antigens. This HIV ELISA simulation experiment has been designed
to detect a hypothetical patient’s circulating IgGs directed at an HIV antigen
(anti-HIV IgG). Several wells will first be coated with simulated HIV. Then a
simulated sample of human plasma will be added to the wells. If the primary
antibody is present, it will bind to HIV. Then a secondary antibody is added,
which can bind to the primary IgG. This secondary antibody (anti-HIV-IgG) is
raised in rabbits and goats and is covalently linked to horseradish peroxidase.
When hydrogen peroxide and aminosalicylate are added to each well, the
peroxidase converts the peroxide to water and oxygen using salicylate as the
hydrogen donor. The oxidized salicylate is brown in color, and indicates a
positive result.
General Instructions:
Work in groups of 4. Record your data and answer questions in order.
Pre-lab: (10 points)
 Define the following: ELISA, immunoglobulin, IgG, primary antibody,
secondary antibody

Answer the following:
1. Describe the entire sequence of immune activation after viral infection,
from being engulfed by an antigen-presenting cell, all the way to
formation of anti-viral IgGs. Use the information from lecture (the
“putting it together” slide). (5 points)
Physiology 3 Lab Manual, Spring 2016
47
Experiment:
1. Obtain a microtiter plate, orient it vertically, and mark the plate with
your group name, and number the rows 1-4.
2. Label 5 transfer pipets as: (-), (+), DS1, DS2, PBS. (-) stands for negative,
(+) stands for positive, DS stands for donor plasma, and PBS stands for
phosphate buffered saline.
3. To all 12 wells, add 100µl of “HIV.” Leave the wells for 5 minutes at
room temperature.
4. Remove all the liquid with a fresh transfer pipet.
5. Wash each well once with PBS buffer by taking the PBS pipette, adding
PBS to the wells (do not overfill), and then using the labeled pipettes to
remove all the liquid from the wells in each row. Dispose of the liquid in
the beaker labeled “waste.”
6. Add 100µl of PBS buffer to the 3 wells in row 1 (negative control, no
IgG). Add 100µl of the “+” (positive control, anti-HIV IgG, primary
antibody) to the 3 wells in row 2. Add 100µl of donor plasma “DS1” to
the 3 wells in row 3. Add 100µl of “DS2” to the 3 wells in row 4.
7. Incubate at 37°C for 15 minutes.
8. Tell instructor to “make the substrate!”
9. Remove all liquid from each well with the appropriately labeled transfer
pipet. Dispose the liquid in the waste container.
10. Wash each well once with PBS buffer as described in step 5.
11. Add 100µl of the anti-HIV-IgG peroxidase conjugate (secondary antibody)
to all 12 wells.
12. Incubate at 37°C for 15 minutes.
13. Remove all liquid from each well with the appropriately labeled transfer
pipet. Dispose the liquid in the waste container.
14. Wash each well once with PBS buffer as described in step 5.
15. Add 100µl of the substrate to all 12 wells.
16. Incubate at 37°C for 5 minutes.
17. Remove the plate for analysis. If color is not fully developed after 5
minutes, incubate at 37°C for a longer period of time.
Questions:
1. 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?
5. Why is the primary antibody screened instead of the virus itself?
Physiology 3 Lab Manual, Spring 2016
6. Why does destruction of T helper cells compromise the entire immune
system?
7. Why is it so hard to make a vaccine for HIV?
8. How would you develop a secondary antibody to test for exposure to
swine flu (a virus)?
9. How would the protocol above differ if you were testing someone for
swine flu?
Disposal
Place all waste in labeled container. Microtiter plates may be thrown in the
garbage.
48
Physiology 3 Lab Manual, Spring 2016
49
Lab 12: 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 antidiuretic hormone released by the posterior pituitary, and activation of
atrial natriuretic peptide and the rennin-angiotensin-aldosterone system. In
this lab you will be designing and executing an experiment to test the effect of
water and Gatorade intake on urine volume.
General Instructions:
There is no pre-lab this week. The entire class will perform this lab on Monday,
and turn in a formal lab report by 8am on Wednesday. For those of you in the
Wednesday lab who cannot attend the Monday section, you may use the data
emailed to you to write a formal lab report, due Wednesday at 8am.
As a class, you will perform an experiment to test the effect of 20 oz. Gatorade
v. 20 oz. water on your urine volume. You will be assigned to either the control
or treatment group. Come to class prepared to perform your experiment by
bringing your assigned drink and adhering to the protocol below. You will write
a formal lab report, as specified near the start of this lab manual. Make sure to
follow all of the instructions for the lab report.
Protocol:
 Before your 8am class, eat a low-salt breakfast and drink 8oz. of water
 No substantial eating or drinking between 9-11:15 am
 11:15 meet in lab
 11:20 urinate and drink assigned fluid (20 oz = 591.4 ml)
 Make a graduated cylinder by pouring 50ml water into a cup and marking
its height, and repeating that process until you reach the top of the cup
 12:30 urinate, measure, dispose of urine in bathroom, and wash hands
Physiology 3 Lab Manual, Spring 2016
50
Questions:
1. What was your hypothesis?
2. What was your treatment group?
3. What was your control group?
4. What was your dependent variable?
5. What is the mechanism by which Gatorade affected the dependent
variable?
6. Why does Gatorade have both salt and sugar in it?
7. If some students didn’t follow the protocol, which kind of bias would this
create? What is negative about this kind of bias (be specific)?
8. If I assigned students who regularly consume Jamba Juice to the water
group and McDonalds fans to the Gatorade group, which kind of bias
would I be creating? What is negative about this kind of bias?
CONGRATULATIONS, YOU HAVE FINISHED ALL OF THE LABS!!!