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Animal Body Systems Laboratory Manual


Copyright ã 2019 by
Tracy Marchant and Sheri Fisher

All Rights Reserved

Students are reminded that they must have an access code to view or print the lab manual.

Cover images from Pixabay (https://creativecommons.org/licenses/by/2.0/legalcode). Reproduced


under Creative Commons.
Original images by:

1. Alinonemovie
2. By OpenStax Anatomy and Physiology -
https://cnx.org/contents/FPtK1zmh@8.25:fEI3C8Ot@10/Preface, CC BY 4.0,
https://commons.wikimedia.org/w/index.php?curid=49891266
3. Patrick J. Lynch
4. Colinoob

Printed in Canada

Copyright 2019 - Tracy Marchant and Sheri Fisher


TABLE OF CONTENTS
General Rules and Organization………………………………………………………………….……………………………… 4

LAB EXERCISE #1
Introduction to Excel, iWorx 214TM and Labscribe 4TM Software1……………………………….……………….. 9

LAB EXERCISE #2
Sensory and Motor System Integration…………………………………………………………………………………….. 21

LAB EXERCISE #3
Recording Nerve Action Potentials………………………………………………………………………………………….… 32

LAB EXERCISE #4
Skeletal Muscle Physiology……………………………………………………………………………………………………….. 42

LAB EXERCISE #5
Osmoregulation………………………………………………………………………………………………………………………… 53

LAB EXERCISE #6
Human Respiratory Physiology……………………………………………………………………………………………..….. 60

LAB EXERCISE #7
Human Circulatory Physiology………………………………………………………………………………………………….. 71

LAB EXERCISE #8
Metabolism………………………………………………………………………………………………………………….………….. 81

APPENDIX 1
Rubric………………………………………………………………………………………………………………………….………….. 90

APPENDIX 2
Sample Figure with Figure Legend and Conclusion……………………………………………. ………….………… 92

1 iWorx 214 and Labscribe 4 are the property of iWorx corporation

Copyright 2019 - Tracy Marchant and Sheri Fisher


BIOL 224: General Rules and Organization 4

General Rules and Organization


The laboratory portion of BIOL 224.3 will contribute a total of 35% towards your final grade in this course.
The overall lab mark will be determined as follows:
Group Assignments (8 X 1%) = 8%
Pre-Lab Quizzes (8 X 0.375%) = 3%
In-Lab Tests (2 X 4.5%) = 9%
Lab Exam = 15%
Total = 35% of final course grade

Missing Labs: Attendance at all regularly scheduled labs, review labs and the lab exam is COMPULSORY
and required for completion of the course. Appropriate documentation will be required for any lab, quiz
or lab exam missed due to illness or extenuating personal circumstances. Notification via e-mail with a
return receipt request to the lab coordinator (Sheri.Fisher@usask.ca) is required within 24 hours of the
missed lab. It is your responsibility to contact us. Missing a lab component without appropriate
documentation and failing to notify the lab coordinator within 24 hours will result in a grade of 0% on
assignments, quizzes, tests and/or the lab exam. Furthermore, students who arrive late or leave before
the assignment is completed may receive a mark deduction on the group assignment. We do not offer
make-up labs. However, if you have a valid prior commitment you may contact the lab coordinator (Sheri
Fisher) and request being able to attend a different lab section within the week. NOTE: We will not
reschedule to accommodate vacation plans. We will not reschedule to allow study time for other courses.
It is your responsibility to arrange alternate test times outside of your regularly scheduled lab.
Lab Partners: You will work in groups of three students. You will be assigned group numbers during the
Organization Lab period. You will not be allowed to switch groups except in special circumstances.
Group Assignments: At the end of each lab exercise, a group assignment will be due. These assignments
are to be composed during the lab period. Late submissions will not be accepted so ensure that your copy
is submitted before the lab period ends. Group assignments involve analysis of experimental data and
the creation of figures in Microsoft Excel. Each figure submitted require a figure legend and short
conclusion in which a physiological explanation is provided. Each of these 8 assignments will be worth
1%. All members should contribute equally to the assignments and as such every member of the group
will be assigned the same grade. It is your responsibility to ensure that all group members have copies of
each group assignment.
Pre-Lab Quizzes: Before each lab there will be an online quiz that must be completed. It will become
available at the end of the previous lab (i.e. the Lab 2 pre-lab quiz will be available after Lab 1 ends). The
quiz will be found through the Blackboard page for your lab section, and can be completed any time up
until 15 minutes before your regularly scheduled lab begins. Questions will be randomized multiple
choice, fill-in-the-blank, or true-false and will be designed to test basic understanding of the lab
procedure. These quizzes are to be completed individually, and feedback is provided to the lab
coordinator and instructors by Blackboard. If anyone is found to be plagiarizing answers, they will receive
a grade of 0% on the quiz and a further reduction of their total lab mark. Repeated infractions, or failure
to complete multiple quizzes, will be brought to the attention of the instructors as Academic Misconduct.
Tests: There will be two lab tests worth 4.5% each that will be written at the start of Lab 4 and Lab 8.
These tests are designed to test your knowledge of previous work done in the lab and will cover
information found within the lab manual. Questions used for the in-lab tests will model the format of the
lab exam. Once the test is complete, you will proceed with the lab experiment.
Lab Exam: The lab exam, worth 15% of your final BIOL 224 mark, will be written during the final lab
period. You will have 1.5 hours to write the lab exam. The questions will be organized into subheadings
based on lab exercises. More information will be given during the review lab.
Access and Equity Services for Students (formerly disability student services): Students who are
registered through AES must inform the lab coordinator and their head lab demonstrator at the beginning
of the term. Students who are registered with AES may be granted accommodations if they self declare
to the lab coordinator and provide appropriate documentation. You must contact the lab coordinator to
make arrangements 3 days in advance of each test. The lab exam does not have a practical component;
contact AES to make arrangements with their office to write the lab exam at least three weeks prior.
Lab Manual & Preparation for the Lab Exercises: At the University of Saskatchewan bookstore you
purchased a unique access code for the digital copy of your lab manual. If you lost your access code
another will have to be purchased. By purchasing the access code you have digital access to the BIOL 224
lab manual and the ability to print one copy of this manual for your own use. You are not permitted to
distribute this manual to others in any form, electronic or otherwise. To do so is considered copyright
infringement and students who do so will be subject to disciplinary action in accordance with University
of Saskatchewan academic conduct policies. Each student registered in Biology 224 must purchase an
access code for the lab manual. Students who fail to do so will be given a 0% on all pre-lab quizzes and
group reports in the lab. The access code is linked to your registration in BIOL 224 and lab manual
purchase will be monitored.
Each student is required to bring either their printed or digital copy of the lab manual to each lab period.
Students are well advised to read the lab description, class notes and the relevant portions of the
textbook BEFORE coming to the lab period. Each lab exercise lists the relevant pages in the textbook. A
section entitled ‘readings from scientific literature’ can be found at the end of each exercise. The relevant
portions of any textbook chapter in these lists are on reserve for this course in the Natural Science Library.
The majority of journal articles can be accessed by clicking on the link provided at the end of the
reference. The readings from the scientific literature generally give more detailed information than the
course textbook and students are encouraged to read these for a greater understanding of the
physiological principle under study. A little advance work on your part will help you maximize your
learning experience during each lab period.

Copyright 2019 - Tracy Marchant and Sheri Fisher


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BIOL 224: General Rules and Organization

Academic Misconduct
The College of Arts and Science has a zero-tolerance policy regarding plagiarism or cheating and instructors
are required to report cases of academic dishonesty to the Dean’s office. Offenders are required to attend a
meeting of the College student discipline committee. If the committee determines you violated the rules of
academic honesty, the typical punishment is a grade of zero on the material, plus a loss of 5-15% on the final
course mark. You will also be unable to withdraw from the course. More severe penalties can be imposed.
For more information on academic misconduct, you are encouraged to visit the following website:
www.usask.ca/honesty.
Important Note: You do not require any additional papers or devices for this lab and must only have the most
current lab manual on your lab bench or computer screen. Bringing in old figures, copied figure legends /
conclusions and old lab manuals from previous years/terms into the lab is considered an attempt to plagiarize
and cheat. If you are caught, you will be instructed to leave the lab room and you will be given a mark of 0%
on that week’s group assignment. When we enact any of these penalties, we report our actions in a letter to
the Dean. This letter will be kept on file and may be used against you if you are ever reported again. Internet
access is restricted to activities specific to the lab (such as emailing lab partners group assignments). You
cannot use the computer for anything but lab related activities while you are in the lab. This means no Google!
Examples of Academic Misconduct: the following represent misconduct cases reported in previous years.
Scenario #1: A group handed in their assignment at the end of the lab period. When the assignment was being
reviewed, it became obvious that the students were not writing about the current experiment but about an
experiment performed in previous years. Inspection of the students’ lab materials showed the students had
copied old figure legends / conclusions into their lab manual. The subcommittee of Student Appeals,
Grievances and Academic Misconduct in the College of Arts and Science determined that the students had
committed an act of academic misconduct. Each guilty student received a grade of 0% on the group assignment
and an additional overall mark deduction of 10%.
Scenario #2: A student was observed with a piece of loose leaf on which was a fully written figure legend /
conclusion and a hand-drawn graph. The figure legend / conclusion contained key words that pertained to an
experiment done in the previous term, rather than the current term. The Subcommittee of Student Appeals,
Grievances and Academic misconduct in the College of Arts and Science determined that the student had
committed an act of academic misconduct. The guilty student received a grade of 0% on the group assignment
and an additional overall mark deduction of 5%.
Don’t Cheat. If you get caught, the penalties are severe. If you cheat, you will not learn material that will be
tested in the quizzes and lab exam. You don’t need to cheat to get a good grade.

Copyright 2019 - Tracy Marchant and Sheri Fisher


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BIOL 224: General Rules and Organization

Lab Rules and Safety Information:


• Turn your cell phones to silent or vibrate during the lab and do not text or make calls during the
lab period. If you need to do this, please be respectful to the other students and do so outside of
the lab room.
• Personal computers or tablets are not permitted. There are double computer monitors in the lab
room, one of which can be used for the lab manual.
• Food and drink, including water, are not permitted in the lab room at any time; any items of this
nature found in the lab will be thrown into the garbage. You can keep bottled water in your bag
but must drink it outside of the lab.
• Dress Code requires you to wear close toed shoes during the lab period
• Personal Protective Equipment (PPE’s) will be required for certain labs. Disposable gloves will be
made available to you for experiments requiring PPE’s. You may wear a lab coat to Lab #5, as you
will be working with samples of Sheep blood.
• Rowdy and obnoxious behaviour will not be tolerated.
• Violation of any of the above rules can result in your expulsion from the lab for the day. You will
not be allowed to make-up the lab. You will receive a grade of 0% on the group assignment.
• Fire or Emergency Evacuation: In the event of a fire or other emergency that requires the lab room
to be vacated, please exit out the nearest lab room door, turn right and proceed out the exit at
the top of the stairs. The fire alarm is tested on the first Monday of every month; you do not need
to exit the building during the test.
• Injury: In the event of an injury, please report the incident immediately to your teaching assistant
who will notify the lab coordinator.
• Lab exercise specific safety information is provided within the experimental write-up.
• Many of the lab exercises will collect non-invasive physiological data from you and your lab
partners. Participation as an “experimental subject” is voluntary. If, for any reason, you do not
want to have your physiological data collected, simply advise your lab partners and if necessary,
your lab demonstrator. The lab exercises involving human subjects are safe and fairly innocuous,
although some routine activities such as mild exercise may be required of volunteers. If you have
any medical condition that might preclude your participation in the lab exercises, please bring
these to the attention of your lab demonstrator. We anticipate (and sincerely hope) that you will
find the various experiments fun and entertaining as well as effective learning experiences. Having
said this, we will not force anyone to do anything that makes him or her uncomfortable. If
necessary, one of your instructors might be convinced to serve as an experimental subject when
there are not enough subjects available in your lab group.
Ethical use of animals in Biology 224: Certain lab experiments will involve the use of invertebrate and
vertebrate animals. Being allowed to use these animals in a teaching lab is a privilege. The animals chosen
for use were done so primarily because of costs and convenience. Furthermore, there are fewer ethical
considerations with invertebrate animals than when using animals with more complex nervous systems

Copyright 2019 - Tracy Marchant and Sheri Fisher


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BIOL 224: General Rules and Organization

and cognitive abilities (i.e. vertebrate animals). However, the basic physiological principles apply whether
we use invertebrates or humans or some other vertebrate species. The animals in the lab are used in
accordance with the guidelines and policies of the Canadian Council on Animal Care (CCAC)
(http://www.ccac.ca/en_/). Additionally, the University Animal Care Committee (UACC) has approved the
use of vertebrate organisms in BIOL 224. Students who plan on taking additional courses that involve
animals can complete the UACC Animal Care Course (http://research.usask.ca/for-
researchers/ethics/uacc-animal-care-course.php). This course is free to U of S students and is
recommended for those BIOL 224 students who might work with vertebrate animals in their future
studies. The BIOL 224 instructors have all completed this course, are committed to the humane treatment
of the vertebrate animals, and to using the minimal number of animals necessary in the teaching of
fundamental principles of animal physiology.
Arts and Science Computer Lab: BIOL 224 labs require the use of Arts and Science computers on a daily
basis. Students from Kinesiology, Agriculture or other colleges often have difficulty logging into the lab
computers and are unable to access the settings folder on the R: drive. Every group member will need
to be able to log on to the computer so ensure you are able to log into the lab computer before you leave
the first lab day.
• Have a group member log into the computer (if no one can, ask a lab demonstrator)
• Go to https://mits.usask.ca
• Log into My Profile
• Beside Password, click on Edit
• Select Sync
• Enter your password and sync password

Copyright 2019 - Tracy Marchant and Sheri Fisher


9
BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

LAB #1: INTRODUCTION TO EXCEL AND


LABSCRIBE
Recommended Textbook Reading:
Sections 44.2e - 44.2f, 45.6 and 50.7 - 50.7d of Russell et al. (2016) Canadian edition
Sections 42.7-42.7d, 45.2a and 45.2f of Russell et al. (2018) Canadian edition

Presenting Scientific Data Using Microsoft Excel:


Microsoft Excel is a spreadsheet program that allows you to sort, filter and analyze data. The chart
capabilities of this program permit you to create scientific figures that concisely represent your data.
In BIOL 224, you will be using Excel weekly to construct figures as a method of data analysis. The two
most common types of figures you will make are XY scatter graphs and column graphs.

XY SCATTER GRAPH EXERCISE

An XY Scatter graph is usually chosen when both sets of data are numerical in nature and on a
continuous scale.

The X stands for X-axis or the scale that runs horizontally across the graph.
The Y stands for Y-axis or the scale that runs vertically up and down the graph.

When asked to plot a XY Scatter, the information will be presented as ‘plot variable 1 versus variable
2’. Variable 1 is the data that will be plotted on the Y-axis, whereas variable 2 will be the data plotted
along the X-axis.

Another way to think of the data is that the data plotted on the Y-axis depends upon the data plotted
on the X-axis. Therefore, the Y variable is the dependent variable and the X variable is the
independent variable. The independent variable can also be the experimental treatment group.

Data to be plotted:
Oxygen is used in the biochemical reactions to generate ATP, a process called aerobic metabolism.
When animals exercise, considerable ATP is used during muscle contraction. Consequently, there will
be a corresponding increase in O2 consumption by the body as cells work to replenish their ATP
stores. Mammals can be trained to walk / run on treadmills while their breathing is monitored
through a respiratory mask. A ground squirrel walking at a speed of 0.4 Km/hr has a rate of oxygen
consumption of 1.7 mL O2/g/hr. At 3.4 km/hr the ground squirrel consumed 3.2 mLO2 /g/hr. At 0.7
km/hr the oxygen consumption was 1.9 mLO2 /g/hr; 2.1 km/hr resulted in an oxygen consumption
rate of 2.5 mL O2 /g/hr and a speed of 1.5 km/hr resulted in an oxygen consumption rate of 2.2
O2/g/hr.

Copyright 2019 - Tracy Marchant and Sheri Fisher


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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

1.) Open Excel. A blank Worksheet should appear.


2.) In the first cell of the left-most column (cell A1) type Running Speed. In the cells below, enter the
running speed (km/hr) in ascending order (lowest to highest).
3.) In the top cell of the second column, (cell B1) type Rate of Oxygen Consumption. Type in the value
for mL02/g/h that corresponds to the running speed in the left-most column.
4.) Using the mouse, highlight this entire data set and click on the Insert pull-down menu. On the
insert page there many functions that are delineated into groupings by a vertical line.

5.) Within the Recommended Charts, select Scatter . You will see that there are 5 sub-types or
options for the scatter graph. Holding the mouse over each of the different graphing options
will allow you to read the explanation for each sub-type, visible in the window below the
sub-types. Select the sub-type that says ‘Scatter with straight lines and markers’.
6.) A figure will appear within the spreadsheet. When you have the figure selected, you will notice
that there are a few additional menu tabs at the top. Chart Tools has two menu tabs
associated with it, Design and Format. When you click on a cell within the spreadsheet you
will notice that the menu items mentioned above disappear. Click on the figure again and
those menu items reappear.
7.) Make sure that the figure is selected. Click on the Design menu tab and select Move Chart and
choose ‘new sheet: Chart1’. Now the figure will appear as a page on its own.
8.) Select Add Chart Elements. This is where the majority of the editing commands are located. Go to
Chart Titles and select none.
9.) Click on axis titles and choose primary horizontal. Observe the textbox that has appeared below
the graph. Either within the textbox or beside the formula bar (ƒχ), type in an appropriate axes
label for the value (X) axis. Repeat those steps for the vertical axis, Remember to include the
measurement units.
10.) Click on the Legend in the Labels grouping and ensure that none is selected. There is no need
for a symbol legend when only one line of data will be present.
11.) Click on the Gridlines menu tab and unselect primary major horizontal and primary major
vertical. These are not necessary.
12.) Some of the graphs you create will result in data that are on the top right side of the figure,
leaving a lot of empty space on the bottom and left hand side of the graph. To eliminate this
wasted space, click on Axes, and on more axis options. A menu should have opened on the
right hand side of the screen. This menu is another way to perform certain editing commands.
You should see Axis options under this you will see Bounds and Minimum. Adjust the
minimum value from 0 to a value that is close to the lowest data value. You may have to play
around with this a bit to find an optimal axis scale.
13.) Repeat this step for the primary vertical axis. Now the data should be located more centrally in
the figure.

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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

14.) Open Microsoft Word and within the Page Setup grouping, click on page layout, select orientation
and choose landscape. Copy your figure from Excel and paste it into Microsoft Word. Resize
your figure to allow room for your figure legend and conclusion. The figure, figure legend and
conclusion should be on the same page.
15.) Type in a figure legend and conclusion for the graph. Remember to include a figure number at
the start of the legend (since this is the first figure it would be “Figure 1:” followed by an
appropriate legend).

Please refer to the rubric in Appendix 1 when creating your figures.

The figure legend and conclusion are vital areas used to convey information within your assignment.
These areas are similar to the way scientific journal articles are presented with separate sections for
introduction, materials and methods, results and a conclusion. Each section requires specific
information within your assignment. The order listed is how they should be covered. For example, do
not give the results before writing about how the data was collected.

Figure legend:
When writing your figure legend there are three main areas that should be covered.

1.) The introductory topic sentence: This is where you introduce the graph type and the relationship
between the variables as named on your axis.

2.) The methods: In this area you indicate the study subject, how was the data collected, the
equipment used. One to two sentences should cover this.

3.) The results: Results are the trend of the data. Be sure to use the variable and / or column names
while explaining the trend. Provide any values or show calculations requested. Two to three
sentences should suffice (of course this is a guideline - some experiments may require only
one sentence, while others may need more).

Conclusion:
Below the figure legend type a separate heading Conclusion. The conclusion is where you endeavor
to explain the physiology behind the results presented in your figure. Explain why and how you
obtained the results using the terminology from the lab manual. If a definition of a term aids in the
explanation, then provide that.
If your data did not match the expected results, indicate that within the conclusion. Then explain
what the results should have been and why.

Hint: If you are writing phrases like 'due to' or 'because of' then you are likely drawing a conclusion
and that information should be in the conclusion, not the figure legend.

Once you have finished this figure, have your lab demonstrator look at it to determine if there are
any obvious errors.

Copyright 2019 - Tracy Marchant and Sheri Fisher


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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

THE IWORX 214 INTERFACE


The iWorx 214 is a peripheral hardware device (Fig. 1) we will be using in all of the lab exercises
for BIOL 224. This hardware collects physiological information as electrical (analog) signals, converts
the analog signals to digital signals, and then sends digital information via a USB connection to the
computer for display and analysis by the LabScribe program.

Figure 1: The front (at top) and rear (at bottom) panels of the iWorx 214.

Inspect the iWorx 214.


Note that the front panel is divided into three sections. The panel at the far left, labeled CH1 &
CH2 Isolated Inputs – Differential Input, will be used next week to demonstrate action potentials
generated by axons in the Cockroach. The centre panel of the iWorx 214 contains four channels
labeled Non Isolated Inputs. The CH1 and CH2 inputs on the center panel connect to other peripheral
devices via a BNC. The CH3 and CH4 inputs connect to peripherals via a DIN8 cable and will be used
to record your pulse data during this lab. The Stimulator and Ground connections are found on the
right panel of the iWorx 214. The stimulator is controlled by the LabScribe program and generates
small output voltages.

THE LabScribe PROGRAM


The LabScribe program controls the parameters associated with the input (i.e. the various
channel connections) and output (i.e. the stimulator) of the iWorx 214. The program also contains
extensive data analysis and presentation functions. In this section, you will learn basic information
about the channel (input) controls in LabScribe. As you go through this section, feel free to click on
control buttons and open dialog windows for inspection. Later on in this lab period, you will conduct
an experiment to give you hands-on practice collecting physiological data with this equipment and
computer program.

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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

Opening LabScribe:
1.) Have one of your group log into to the Arts & Science computer.
2.) Turn on the power to the iWorx 214 using the switch on the rear panel (Figure 1). The
iWorx box has to be turned on prior to opening LabScribe.
3.) Double click on the LabScribe icon located on the desktop.
4.) A pop-up window may appear - click Teaching.
5.) A second pop-up may open requesting language selection - click Default Language.
6.) The program will open and a pop-up window will appear indicating that the iWorx 214
hardware was found - click OK.
7.) If the LabScribe icon is not present on the desktop, go to the Start Menu, select All
Programs, go to the iWorx Folder and select LabScribe2.
Using the LabScribe Program:
When LabScribe is opened you will notice that many of the top Menu Bar controls are similar
to other software programs, including File, Edit, View, Tools, Settings and Help. We will explore the
main menu controls more thoroughly during the acquisition and analysis of data later in this section.

When LabScribe is first started, it will always open to the settings that were last used.
Reset the software by selecting Settings from the top menu bar and select Default Settings.

The main LabScribe window should now look as shown in Figure 2. The channels display data
along two axes. The y-axis represents the strength of the input signal data collected by the iWorx
214. The x-axis represents the time frame over which the signal is to be recorded. This setting is
shown as the Display Time in the upper left. The two vertical red lines show the current location of
the two Cursors. These cursors will be used later to measure various aspects of the input signal (your
pulse) with these measurements appearing in the upper right of the window. In Figure 2, the distance
between the two cursors measured on the x-axis is shown in the upper right as T2-T1 = 30.00 msec.
Note that time (i.e. the x-axis) is common to all channels. The distance between the two cursors as
measured on the y-axis appear in the upper right corner of each channel as V2-V1 = 0.000 volts. The
red Record button in the upper right is used to initiate a data recording. Other controls and displays
in the main LabScribe window will be explained as we proceed through this exercise.

Figure 2: Appearance of the main LabScribe


window in the default setting.

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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

Below the main menu bar at the very top of the main LabScribe window is a second tool bar
with the following controls:

The general functions of these LabScribe ToolBar controls are summarized below. Additional
explanations of the various controls will be provided as needed later in this exercise.

New File: Opens a new file. Only one acquisition window may be open at a time.

Open File: Opens a previously recorded file.

Save File: Saves data to the file currently open.

Main Window: Brings the Main window to the foreground.

Analysis Window: Brings the Analysis window to the foreground.

XY View: Brings the XY View window to the foreground.

FFT (Spectrum): Brings the FFT window to the foreground.

Journal: Opens the Journal in the right hand side of the window.
Meter: Displays or hides the meter to the left of the channel windows

Marks: Brings the marks list to the foreground.

Stimulator Preferences: Hides or displays the Stimulator toolbar.

Half Display Time: Reduces the time displayed on the screen by a factor of ½ each time
the icon is clicked.

Zoom Between Cursors: Zooms to the area selected by the two cursors.

Double Display Time: Increases the time displayed on the screen by a factor of 2 each
time the icon is clicked.

Autoscale All: autoscales all the channels in the main window.

Double Cursors Mode: Click icon to display two cursors on the data window. Time and
voltage differences between the data points intersected by the cursors are measured.

Single Cursor Mode: Click icon to display one cursor on the data window. Absolute
time and voltage from the beginning of the trace to the cursor are displayed.

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BIOL 224: Introduction to Excel, iWorx 214TM and Labscribe 4TM.

The information above is a very brief overview of some of the functions of the iWorx 214 and
LabScribe program. There are many other capabilities within the program that will be explained as
we move through this and other lab exercises. The following experiment is designed to provide you
with experience recording data, as well as analyzing and presenting data.

EXPERIMENT 1: EFFECT OF COOLING ON PERIPHERAL BLOOD FLOW


Depolarization of the myocardial cells in the ventricle causes the ventricles to contract and
force blood into the major arteries of the circulatory system in a pulsatile manner. The pulses of blood
moving in arteries can be recorded using a device known as a plethysmograph. In this experiment,
you will record the pulse wave in the finger of human volunteers (i.e. you and your lab partners). You
will examine the effects of temperature on peripheral circulation.
Located within the dermis there are specialized sensory receptors that are sensitive to
changes in temperature. Once activated, these peripheral thermoreceptors send signals to the
hypothalamus that in turn causes a physiological change that includes an alteration in blood flow. The
volume of blood traveling through arteries can be increased or decreased by altering the diameter of
those arteries. When arteries located in peripheral tissues undergo vasoconstriction there is less
blood supplied to the tissue and a decrease in pulse amplitude is observed as a result of the decrease
in peripheral blood flow. Conversely, vasodilation of peripheral arteries results in an increase in
peripheral blood flow and an increase in the amount of blood supplied to those tissues.
Equipment Required
PC Computer
iWorx/214 data acquisition unit & power supply
USB cable
PT-104 pulse plethysmograph
Ice and a plastic bag
Loading the Settings File
These steps will automatically configure the settings within the LabScribe channel for the
experiment.
1.) In LabScribe, go to the Settings pull-down menu at the top of the screen.
2.) Select Load-group.
3.) A pop-up window entitled Choose a file will appear.
4.) Click the My Computer icon on the left hand side and select the Shared Drive R:
5.) Double click on the Biology 224 folder.
6.) Click once on the file Biol224.iwxgrp and click Open at the bottom of the window.
7.) Within LabScribe under Settings you should now see a folder called Biology 224.
Select this and open the file called pulse.
Equipment Set-Up and Functional Check
1.) Locate the PT-104 pulse plethysmograph on the lab bench.

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2.) Plug the DIN8 connector of the PT-104 into the Channel 3 Non-Isolated input of the iWorx
214 interface (Figure 3).
3.) Wrap the plethysmograph firmly around the end of the volunteer’s middle finger with the
finger tip placed on the white pad. Have the volunteer sit quietly with their hands
(palms face up) on the bench in front of them. Movement during the pulse recording
will result in erratic recordings that cannot be used for data analysis.
4.) Fill the plastic bag 2/3 full with ice from the cooler and return to your lab bench.
5.) To begin sampling click Record; you will notice that the Record button has turned into a
Stop button.
6.) Click on the AutoScale button at the upper margin of the Human Pulse channel and
the Double Display Time button below the main menu bar. These two buttons are
very useful when you need scale your data to fit the channel window. Your recording
should look like Figure 4.
7.) If the signal on the Pulse channel is upside down when compared to trace in Figure 4,
click on the downward arrow to the left of the channel title and select the Invert
function. Alternatively, you can right click on the channel window and select Invert.
The trace should now look similar to Figure 4. If the pulse signal is small or noisy, stop
recording and adjust the tension on the strap holding the pulse plethysmograph to the
finger. Make sure you have a good recording before you proceed.

Figure 3. The PT-104 pulse plethysmograph (left) and the plethysmograph with the DIN8 connector
plugged into the Non-Isolated Input channel 3 (right).

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Mark box
Downward arrow

Channel
window

Figure 4: A typical human finger pulse recording in LabScribe.

Collecting Experimental Data


1.) Click on Record.
2.) Keeping the mouse cursor over the mark box type the volunteer’s initials and PRE-
COOL into the markbox next to the mark button and hit enter on the computer
keyboard.

3.) Continue to record continuously for about 2 minutes (you will see the time on the top
right hand side of the LabScribe window right next to the record/stop button).
4.) Place the bag of ice lengthwise on the volunteers forearm.
5.) Type COOL into the mark box and click enter.
6.) Record for 3 minutes.
7.) Remove the bag of ice.
8.) Type REWARM into the mark box and hit enter.
9.) Continue to record for 3 minutes.
10.) Stop recording and go to the File menu and click Save as, give the file an appropriate
name and save it to your V: drive. DO NOT ATTEMPT TO SAVE TO THE R: DRIVE AS
THIS WILL CAUSE THE COMPUTER TO CRASH.
11.) Keep the LabScribe file open.

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Data Analysis in LabScribe: You will be measuring the height (amplitude) of the pulse
waves.
1.) At the bottom of the LabScribe window is a blue scroll bar that allows you to move
through the collected data. Scroll to the start of the pulse data and look for 5 strong
successive pulses. It may be useful to use the Double Display Time button to get a
better view of your data.

2.) Click on the Double Cursors mode button and place the cursors on either side of the
5 pulses and then click on the Zoom Between Cursors button .
3.) Click on the Double Cursors mode button.
4.) Click on the analysis window.
5.) Click on add function and pull it down to V2-V1.
6.) Place one cursor at the onset of the pulse (just before the wave begins to incline) and the
second cursor on the pulse peak. The V2-V1 box now shows you the Pulse Amplitude.
7.) Go to the Tool Menu along to top of the screen and choose Add all data to Journal. Open
the Journal and you should see Pulse Amplitude and the associated height of V2-
V1. To organize your data in the Journal, include a heading that includes the volunteers
name and the treatment type.
8.) Scroll through to the second deflection and perform steps 6 and 7 for each of the pulses.
9.) Remember to Save!
10.) Calculate the average (mean) amplitude in volts (or millivolts) for this experiment. The
average value of these 5 pulses will be used for the PRE-COOL period pulse amplitude
data. Hint: you can copy and paste the data from your journal into excel and using the
average function to perform your calculation. In Excel highlight the data of interest,
click on the arrow beside the sum symbol S, and select average.
11.) Unzoom the data by clicking Double Display Time.
12.) Scroll through the data until you come to the COOL mark. You can also click on the Marks
icon in the LabScribe toolbar. A pop-up window will appear allowing you to view
the marks you entered during the exercise. By clicking on the number along the left
hand side the mark information will become highlighted. When the mark information
is highlighted you can select Go To Mark and the program will automatically position
the recorded data at the time of the mark into the main window.
13.) Once you have found the COOL mark, scroll through your data until you find the data
which shows the maximum response to the treatment.
14.) Repeat steps 6 and 7 above to find the average of 5 successive pulses for the COOL period
pulse amplitude data.

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15.) Repeat the above procedures for 5 successive pulses of the REWARM data taking
your measurements near the end of the recording period.
16.) Save the file.

Excel Analysis and Graphical Presentation:


COLUMN GRAPH
A column graph is appropriate when the independent variables are non-numerical categories such as
treatment group. The independent variable categories are displayed along the x-axis. The y-axis for
the dependent variable will have a numerical scale. A column graph can also allow you to show more
than one series of data in the graph. For example, when the dependent variable data is presented for
each sex.
1.) Open the Microsoft Excel program.
2.) Use the averages from your data set (PRE-COOL, COOL & REWARM) to create a
column graph.
3.) Enter the treatment names in cells A1, B1 and C1 in the Excel spreadsheet and the
associated amplitude in the cells immediately below.
4.) Highlight all the information typed into the cells and click on the menu tab Insert and
select column which is grouped with the other chart types. There are many column
graph options, you will select the upper leftmost graph which is a clustered 2D
column.
5.) Follow the instructions given for the XY scatter graph for moving the chart to a new
sheet, adding axis titles, etcetera.
6.) Copy and paste the figure into Microsoft Word.
7.) Write an appropriate figure legend and conclusion as described in the XY Scatter Plot.

Check with your lab demonstrator to ensure that you have successfully completed the figure.
Be sure to include all names and Student Identification # (SID#) from your lab group members, your
group #, and the lab day and time on the first assignment page in a textbox.
Digital Hand-in Process:
All assignments will be handed in using Blackboard. At the end of each lab, one group member
must sign into PAWS and follow the instructions below to save your assignment as a PDF and submit
for grading.
1.) Copy the column graph, figure legend and conclusion in to a single Microsoft Word file. Double
check formatting to ensure that nothing is cut off or missing.
2.) Insert a text-box on the top right corner of the first page. Write the names and SID# of
your lab group members, your group #, and the lab day and time.
3.) Open the File drop down menu and select Save As…

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4.) Change the Format to PDF.


5.) Name the file using the group number and the last names of your group members in the
format Group##_Lab##_LastName1LastName2LastName3.pdf. This naming convention is
necessary so that files, and marks, are properly attributed. Incorrect file names will result in
lost marks.
6.) Have any group member log in to PAWS.
7.) Select Course Tools from the list on the left (this may require opening the menu by clicking on
the icon in the top left corner first).
8.) Click on My Courses and select your BIOL lab section (Biol-224-LX, where X is the number of
your section.
9.) From the menu on the left choose Course Materials.
10.) Click on the group assignment for the week that has your Lab Demonstrators name (ex. Lab 1
Rachel).
11.) In the second section of the submission form, select Browse My Computer for copyright
cleared file.
12.) Select the correctly named PDF file.
13.) Leave all other boxes blank.
14.) Click the green Submit button at either the top or bottom right.

Group Assignment Due Today:


Figure 1: a column graph of pulse amplitude versus PRE-COOL, COOL & REWARM treatments.
Convert graphs in to a single PDF file and hand in using Blackboard. Name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.pdf
Be sure to include all names and Student ID# (not NSID) from your lab group members, your
group #, and the lab day and time on the first assignment page.

Question to ponder: When analyzing your data, you were asked to find the maximum
response to the ice treatment. Why does the time to the period of maximum response differ for
each individual?

Readings from The Scientific Literature: (on reserve in the Natural Sciences Library)
Downey, J.A. 1968. The effects of heat, cold, and exercise on the peripheral circulation. Archives
of Physical Medicine and Rehabilitation 49: 308 -14.
Insler, S.R. & M.D. Sessler. 2006. Perioperative Thermoregulation and Temperature Monitoring.
Anesthesiology Clinics 24:823-837.

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Biol 224: Sensory & Motor System Integration 21

LAB EXERCISE #2: SENSORY & MOTOR


SYSTEM INTEGRATION
Recommended Textbook Reading:
Sections 45.3, 45.4 and 45.8 of Russell et al. (2016) Canadian edition
Sections 45.2c, 45.2d, 45.3a of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION
Animals continually monitor changes in the environment and react appropriately. A stimulus
from the internal or external environment is detected by one or more sensory receptors (neurons),
which send this sensory information to the central nervous system (CNS) for processing. Next week,
action potentials (APs) from a cockroach leg stretch receptor will be recorded. This week we will
conduct experiments to further investigate the role of the sensory nervous system and specifically
how sensory information is integrated by the CNS in human volunteers. Nervous system integration
can be simply defined as the generation of a motor output based on the sum of the sensory inputs.
Much of the integration within a nervous system is done by way of reflex arcs. A simple reflex
arc, involving relatively few sensory and motor neurons, is an easy way to learn about the stimulus-
response relationship. A loud sound or something flying at your eye makes you blink while a tap on
the tendon under the kneecap produces the involuntary patellar (knee-jerk) reflex. The neuronal
circuitry required for the patellar reflex is confined to the spinal cord. Sensory information also
ascends to higher centers, but the brain is not required to perform the reflex. More complex reflexes
usually involve additional interneurons and more than one population of motor neurons. Simple
reflexes can send information very quickly; as more neurons and synapses are involved, there is a
longer delay between the stimulus and response.
Although much of the integration within the CNS is done by way of simple or complex reflex
arcs, learning can also be used to modify motor outputs. Brains are usually the location where
learning takes place. The neuronal and physiological mechanisms underlying learning are complex
and not fully understood, and are areas of active research today. However, learning to respond
appropriately to new environmental stimuli is exceedingly important. Animals with larger brains are
able to learn more quickly and to generate more complex responses. This is primarily due to the
presence of more interneurons and neuronal connections, which, in turn, allow for more complex
patterns of integration.
In this lab exercise, you will study visual reaction times in human volunteers. The time from
the start of a signal to the response is termed reaction time. Many factors affect reaction time
including: signal complexity, duration and strength. Furthermore, there are three ways in which the
sensory system stimulated affects reaction time. First, there are differences in afferent conduction
times between sensory systems. Second, some sensory systems can change instantly while others
change more slowly. Third, certain sensory systems are more sensitive. Reactions are voluntary
responses that can be modified through learning.
During this lab you will also conduct a simple experiment to measure the rate at which learning
takes place in a volunteer. The process of increasing the accuracy, speed and coordination of tasks

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Biol 224: Sensory & Motor System Integration 22

involving eye-hand coordination the more often we perform them is called visual-motor learning.
Visual-motor learning is one of the most important adaptive processes that our bodies possess. It
allows us to modify our behaviours in response to new and changing visual environments and to
improve our performance on tasks that we do repeatedly.
Equipment Required
PC Computer and iWorx 214 data acquisition unit
EM-100 Event Marker
Ear plugs
Prism goggles
Modelling clay to form 5 balls approximately 2.5 cm in diameter
Three differently coloured markers
Tape measure
Large sheet of paper
EXPERIMENT 1: REACTION TIMES
For this experiment, have one student sit directly in front of the computer screen with a finger
from their dominant hand over the Enter key on the computer keyboard, while the other student
positions their foot over the Foot Reaction Switch. The student at the keyboard will hit the enter key
which will generate a vertical line through the LabScribe data window which is the visual cue. During
auditory trials, the sound of the keyboard click will be the auditory cue. As soon as the student sees
the vertical line they will depress the Event Marker creating an upwards deflection. The student
should wear ear plugs to ensure that he/she cannot hear the sound generated when the enter key
was pushed on the keyboard. There should be five recordings of reaction times to a visual cue. Adjust
the monitor so that the study subject can no longer see the screen. They should also remove their
ear plugs and obtain five recordings of reaction times to an auditory cue. The student at the
computer should wait between five and ten seconds between each time they press enter to vary the
time between cues to negate an anticipatory response of the study subject.
Equipment Setup
1.) Turn on iWorx 214 and open the LabScribe program. No device found? Tools, find
hardware.
2.) On the main window, pull down the Settings menu and select the Biology 224 folder. No
settings folder found? See Lab 1, page 15.
3.) Open the settings file entitled A.V.Reaction.
4.) Locate the EM-100 event marker (Figure 1).

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Biol 224: Sensory & Motor System Integration 23

Figure 1. The EM-100 Event Marker & FRS-100 Foot Reaction Switch.
5.) Plug the DIN8 of the EM-100 into Channel 3 input of the iWorx214.
6.) Click on the Record button in the LabScribe program.

7.) To begin, the study subject should wear ear plugs and the student at the keyboard should
quietly press enter AND the student with the Event Marker should depress the switch as
soon as they see the vertical deflection on the computer monitor. A vertical line should
be visible when enter is hit. If no line is present, make sure your mouse cursor is hovering
above the mark box when you press enter or you may need to place a period before enter
is hit.
8.) Repeat step 7.) until you have 5 visual reaction times
9.) To collect the 5 auditory reaction times to an auditory cue, the study subject should
remove their ear plugs, turn away from the computer screen and student at the
keyboard should loudly press enter. The student with the Event Marker will depress the
switch as soon as they hear the keyboard click.
10.) At the end of the 10 trials (5 visual and 5 auditory), click Stop and save the file on your
V drive as B224 ReactionTimes.iwxdata.

Data Analysis and Presentation:


1.) Go to the beginning of your data.
2.) Click on the analysis window.
3.) Click on add function and pull it down to T2-T1.

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Biol 224: Sensory & Motor System Integration 24

4.) Place one cursor at the onset of the cue (the black line created when enter was hit on the
keyboard) and the second cursor at the beginning of the upward deflection created
when the foot reaction switch was depressed.
5.) Go to the Tool Menu along to top of the screen and choose Add channel data to Journal.
Open the Journal and you should see Reaction Time and the associated time of T2-T1.
6.) Measure the reaction times for each trial (5 visual and 5 auditory) for the student.
7.) Calculate the average (mean) visual and auditory reaction time in milliseconds.
8.) Give the mean reaction times to your lab demonstrator. We will compile data from the lab
and provide you with class data.
9.) You will be provided with class data as an Excel file similar to the data below.

visual auditory
300 250
280 220
250 225
290 200
400 280
410 350
305 190
350 210
280 305
265 175
Mean 313 240.5
standard deviation 55.34 55.65
confidence interval 34.30 34.49
lower CI 278.70 206.01
upper CI 347.30 274.99
standard error 0.00 0.00
standard error 17.50 17.60

11.) Find the class averages (mean) for each cue type. In Excel highlight the data of interest,
click on the arrow beside the sum symbol S, and select average.
12.) Determine the standard deviation for each cue type. A few cells below the average type
=STDEVS. A cursor will be within the parenthesis, to add data within the parentheses
highlight the same values you used for finding the average (do not include the average
when highlighting data). The standard deviation must be calculated to determine both
the confidence intervals and standard error of the mean.
13.) For each cue type find the 95% confidence interval. In an empty cell type
=confidence.norm. The cursor will be within parenthesis. Three values must be entered
within the parentheses; alpha = 0.05, standard deviation is the value that was

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Biol 224: Sensory & Motor System Integration 25

determined in step 12, and size is how many students (n) contributed to the class data.
14.) Calculate the upper and lower confidence levels for each cue type. To each reaction time
class average, add the confidence interval to find the upper confidence limit and then
subtract the confidence interval to find the lower confidence limit. Present the confidence
levels in your figure legend.
15.) Find the standard error of the mean (SEM). There is not a built-in formula for SEM within
excel. The formula is SEM = (STDEVS / √n). To find SEM using Excel type = and click on
the standard deviation you determined earlier. After the standard deviation value enter
the divide symbol / and type SQRT and a set of parentheses. Within the parentheses enter
the n value (the number of students that provided data). You cannot calculate a
standard error for the student data. However, when you add the SEM bars a value needs
to be assigned for the student standard error. Therefore, you will need to enter 0 into the
spreadsheet as was done with the data above.
16.) Create a Microsoft Excel column graph to compare the average reaction times of your
group member to the class average for the visual and auditory reaction times. Click on
the + sign at the bottom of your spread sheet to open a new sheet. Leave Cell A1
blank. In cell A2 type student, in cell A3 type class. In cell B1 type Visual and in C1 type
Auditory. Enter the average reaction times. Highlight all cells A1 to C3 and create your
column graph.
17.) Add SEM bars to the columns of data. Right click on your graph, select data, switch
rows/columns. Click on the visual column and choose ‘chart layout’ on the top of Excel.
Select errors bars, choose error bars options, select custom and specify value. A pop-up
window will appear called ‘custom error bars’. In both the positive and negative error
value click on the cell within the spreadsheet for the SEM of visual reaction time data.
Repeat these steps for the auditory reaction time data.
18.) You will notice that SEM bars were added to the student data. These need to be removed.
To do so, right click on the error bar and select format error bar. Choose the end style,
no cap.
Understanding the Statistics:

Statistics is an important method for analyzing and interpreting data. The statistical analyses
performed above will aid in the understanding of the data collected today. Since every student is
different, the physiological data collected from a group of students is inherently variable. The ability
to determine whether any individual result lies within the normal range is critical for comprehending
scientific data. The SEM quantifies how much the reaction times vary amongst the students. The
confidence intervals help answer the question ‘how sure are you that your data is within the normal
range?’. A 95% confidence interval represents the range of values that you can expect to contain
the true mean of the population 19 times out of 20 (i.e. 95% of the time). The SEM indicates how
precisely the calculated mean clusters around the mean of the entire population. A smaller SEM
signifies a smaller amount of variation in the population as a whole.

Scientists use various statistical tests to determine if the differences they observe in their data are

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Biol 224: Sensory & Motor System Integration 26

statistically significant (e.g. the result would not be observed 95% of the time) or just due to random
variation in a population (e.g. the result would be observed 95% of the time). An explanation of
these statistical tests is beyond the scope of this course, but there is a quick rule of thumb that can
be followed. If the data you obtain lies within the 95% confidence interval calculated for class data,
your data will not likely be statistically different from the population as a whole. Another way to
quickly assess your data is to look at the SEM calculated for two groups of data. The data will likely
be statistically significant if there is no overlap in the SEM between the two groups. In other words,
the data is being influenced by more than just random variation in a population. In physiology, this
non-random influence will most likely be due to a specific treatment or some other population
variable we are studying.

Most programs in the biological sciences require students to complete formal courses in statistics
that will provide a much more comprehensive explanation of all things statistical. The instructors of
BIOL 224 strongly encourage students to complete one or more courses in basic statistics early in
their degree program. This will allow students to learn advanced material more easily and with a
greater depth of understanding. Some course options available to you are STAT 245, STAT 246 or
PLSC 213. Sign up for one of these (or another recommended by your program area) next term!

EXPERIMENT 2: VISUAL-MOTOR LEARNING


In this experiment, a volunteer will perform a skill that requires eye-hand coordination
(throwing a ball). The ball throw will also be performed while the volunteer is wearing prism goggles
that cause a shift in the visual field. This will cause difficulties in the ability to throw accurately
because objects in the environment will appear shifted in space. Visual-motor learning will take place
and, after practice, the volunteer will be able to throw accurately while wearing the prism goggles.
Your task in this lab will be to quantify how this visual-motor learning takes place.
A prism is commonly a wedge-shaped piece of glass or other similar translucent material. In
addition to breaking light into its component colours, a prism bends the path of light passing through
it. Because light travels more slowly through glass than through air, it is bent when it passes from
the air to the glass and again from the glass to the air. The amount that light is bent is based on the
index of refraction of the materials through which it passes.
When light passes through a prism, the path of the light is always bent toward the base (thick
end) of the prism. Because of this, a person viewing the world through prisms sees that the world is
“shifted” in space. In this experiment, the prisms placed in the goggles shift the visual environment
in a vertictal direction. Thus, individuals wearing prism goggles will have to look up or down
(depending on how the prisms are mounted) in order to see a target directly in front of them.

This experiment requires at least three people. Divide your group members as follows:
Thrower - throw the ball at the target
Recorder - mark where the ball hits the paper
Ball giver - hand the thrower the balls

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Biol 224: Sensory & Motor System Integration 27

Part 1: Normal Throws Without Goggles (throws 1 – 10)

1.) Determine what role each person will undertake. Draw a vertical and horizontal line
across the paper and clearly mark a target at the cross section of those lines (see
Figure 2). Tape the paper to the wall as instructed by your lab demonstrator.

2.) The Thrower will throw 10 times for this phase. These throws serve as a control or
baseline throws as no manipulation has been performed on the thrower. It is
important to be consistent, so the throws must occur at a fairly regular interval (about
every 5-10 seconds). Avoid taking too much or too little time between throws.

3.) The Ball giver should hand the thrower a ball in the thrower’s dominant throwing hand
(i.e. the hand that is normally used for throwing). Use the modelling clay to form at
least 5 balls of approximately the same shape and diameter (about 2.5 cm). If
necessary, reform the ball after it has been thrown and before it is returned to the
thrower.

4.) The thrower should throw the ball overhand at the target. The thrower should look
directly at the target and not at his/her hand. The thrower does not need to throw
with force- rips in the paper will cause problems with data analysis.

5.) After each ball hits the paper, the recorder should mark the spot where the ball hit with
an X and number (Figure 2). The number should correspond to the number of the
throw 1 through 10. Use the same coloured marker for each of the control throws.

Hints: Avoid letting too much time pass between throws. A good throwing rate is just enough time
to allow the recorder to mark the throw. Also, the recorder should make small marks—big enough
to read, but small enough so that total number of 45 marks can be placed on the board at the same
time.

Part 2: Throwing With Prism Goggles (throws 11 to 30)

1.) Place the prism goggles on the thrower and quickly have the thrower resume
throwing with the exact same procedure as described for Part 1, except complete 20
throws.

2.) Use a different coloured marker to number these throws 11 to 30.

3.) As before, it is important that the thrower looks directly at the target and throws where
the target appears. The thrower should not try to create a secondary strategy to
overcome the prism goggles.

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Biol 224: Sensory & Motor System Integration 28

Part 3: Return to Throwing without Goggles (throws 31 to 45)

1.) Remove the goggles from the thrower and quickly have them throw 15 more balls.

2.) Mark the spots where the balls hit by using the third coloured marker. Number these
throws 31 through 45.

Data Analysis

The basic data are 45 ball-strike locations marked on a large piece of paper. This data has to be
transformed into a set of numerical values that can be used to more closely examine the behaviour.
For each throw indicated below, you will record one value. You will be determining the averages for
sets of throws.

Throws 5 to 10 (Control Throws)


Throws 11 to 15 (Early Throws With Goggles)
Throws 26 to 30 (Later Throws With Goggles)
Throws 31 to 35 (Early Throws After Goggles)
Throws 41 to 45 (Later Throws After Goggles)

1.) Using the measuring tape, mark out 2 cm intervals along the vertical line.

2.) Open a Microsoft Excel worksheet and in cell A1 type Throw Number. To finish this
column of data enter the required throws in the cells below.

3.) In cell B1 type Vertical Displacement.

4.) As shown in Figure 2, X and Y represent Horizontal Displacement and Vertical


Displacement. The horizontal displacement (X) is obtained by measuring the distance
between the mark on the paper and the vertical line. The Vertical Displacement (Y) is
obtained by measuring the shortest distance between the mark on the paper and the
horizontal line. This value quantifies how far above or below the throw landed from
the target. If the mark is above of the horizontal line, make it positive. If the mark is
to below, make it negative.

5.) One person should measure the distances of all the throws to minimize discrepancy
between individuals. Measure the throws in numerical order and give the person
typing on the computer the value for vertical displacement (remember to tell them if
it is positive or negative).

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Biol 224: Sensory & Motor System Integration 29

-x,+y +x,+y

7
3
6 1
5

10
4
vertical
displacement
2
8
horizontal
9
displacement
-x,-y +x,-y

Figure 2. Quantification of Individual Thrower Data. This schematic representation shows recorded
throws on a large piece of paper. The cross in the middle represents the target and the numbers the
successive throws. To transform the data in to numeric values, the locations of the throws are
recorded as X,Y-coordinates relative to the target. X-coordinates are measured as the distance (in
cm) the throw is away from the vertical line. Y-coordinates are measured as the distance the throw
away is from the horizontal line.

Data Presentation:
1.) Use a calculator function of Excel (highlight the throws of interest, click on the arrow
beside the sum symbol S, and select average) to determine the average vertical
displacement for the following sets of throws:
Throws 5 to 10 (Control Throws)
Throws 11 to 15 (Early Throws With Goggles)
Throws 26 to 30 (Later Throws With Goggles)
Throws 31 to 35 (Early Throws After Goggles)
Throws 41 to 45 (Later Throws After Goggles)
2.) Draw an Excel column graph of the average vertical displacement for the five sets of
throws indicated in point 2 above.

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Biol 224: Sensory & Motor System Integration 30

Group Assignment Due Today:


1.) An Excel column graph comparing speed of visual and auditory reactions between an
individual and the class average. Be sure to include the 95% confidence levels calculated
in the figure legend.
2.) An Excel column graph of average vertical displacement during the various throws.
* Remember all graphs require a figure legend and conclusion

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to
include your group number, lab day, all names (first and last) and Student ID# from your lab
group members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3. Please note that Lab## refers to the
weekly experiment, not the lab section.

Question to ponder: What are the major differences between a reaction and a reflex?
Think about whether you would you expect one to result in a faster response time? Would
you expect to observe an improvement in response time with repetition?

Readings From The Scientific Literature: (Through the link below or on reserve in the Natural Sciences
Library)
Almirall, H. and E. Gutiérrez. 1987. Auditory and visual reaction time in adults during long
performance. Perceptual and Motor Skills. 65: 543-552
Blasquez, P., Y. Hirata, and S.M. Highstein. 2004. The vestibulo-ocular reflex as a model system
for motor learning: what is the role of the cerebellum? The Cerebellum 3: 188-192.
Click here for download
McEwen, B.S, and A.M. Magarinos 2001. Stress and hippocampal plasticity: implications for the
pathophysiology of affective disorders. Human Psychopharmacology 16: S7-S19.
Click here for download
Michel, J., H.J.A. van Hedel and V. Dietz. 2007. Facilitation of spinal reflexes assists performing but
not learning an obstacle-avoidance locomotor task. European Journal of Neuroscience. 26:
1299-1306. Click here for download

Pfister, M., J-C L. Lue, F.R. Stefanini, P. Falabella, L. Dustin, M.J. Koss, and M.S. Humayun 2014.
Comparison of Reaction Response Time between Hand and Foot Controlled Devices in
Simulated Microsurgical Testing. BioMed Research International v2014 Article ID 769296 8
pages.
Rosenbaum, D.A. 2010. Reaching and Grasping. In Human Motor Control. pp. 211-250.
Amsterdam. Elsevier Inc.

Copyright 2019- Tracy Marchant & Sheri Fisher


Biol 224: Sensory & Motor System Integration 31

Schmidt, R.A. and C.A. Wrisberg. 2008. Processing Information and making decisions. In Motor
Learning and Performance pp. 25-57. Champaign, IL. Human Kinetics.
Teyler, T.J., J.P. Hamm, W.C. Clapp, B.W. Johnson, M.C. Corballis and I.J. Kirk. 2005. Long-term
potentiation of human visual evoked responses. European Journal of Neuroscience. 21:
2045–2050. Click here for download

Copyright 2019- Tracy Marchant & Sheri Fisher


Biol 224: Recording Nerve Action Potentials 32

LAB EXERCISE #3: RECORDING NERVE


ACTION POTENTIALS
Recommended Textbook Reading:
Chapter 44 and sections 45 – 45.2d of Russell et al. (2016) Canadian edition
Chapter 45 but specifically sections 45-45.2b of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION
Mechanoreceptors are specialized sensory nerve cells that respond to touch, pressure,
sound, movement and stretch. Mechanoreceptors can detect both external and internal stimuli.
External stimuli include vibrations or touches that are created by wind, sound, water, or those that
are transmitted through the substrate. The internal mechanoreceptors function to provide
information about muscle position, contraction, movement as well as leg positioning in space and
are referred to as proprioreceptors. Each mechanoreceptor is associated with a mechanoreceptor
neuron. When a mechanical force (e.g. movement) stimulates a mechanoreceptor, an action
potential is triggered in the mechanoreceptor neuron that travels to higher ganglion for processing.
Cockroaches (Periplaneta americana) have a number of different kinds of mechanoreceptors
on the cuticle, or exoskeleton, of their legs. These mechanoreceptors include: 1) short hairs called
setae, 2) clusters of hairs called hair plates, which bend when adjoining surfaces of the cuticle
contact each other in movement, 3) dome-like structures called campaniform sensilla, which are
distorted with the movement of the spines that protrude from the surface of the sensilla, or with the
movements of the leg, and 4) structures under the cuticle called chordotonal organs, which change
in length when the joint that the organ spans is extended or flexed.
In the cockroach, the neuronal axons associated with the receptors on the tibia travel through
a large sensory nerve that passes through the cockroach’s femur. Individual action potentials are
easily recorded through the use of extracellular needle electrodes placed along this sensory nerve.
The iWorx 214 hardware and LabScribe program will be used in the experiment of this lab exercise
to record and display actual nerve action potentials (APs) from axons of the sensory neuron in the
cockroach leg. Stretch receptors (such as chordotonal organs) are used to detect the position of the
leg as the cockroach moves. The stretch receptor synapses with interneurons in the cockroach’s
central nervous system (CNS) and forms part of a complex neural reflex pathway that helps to
coordinate cockroach movement. As the leg is moved from a neutral position, the sensory neuron
increases the rate at which it generates APs.
This change in firing rate (action potentials/second) is used within the cockroach’s CNS to
monitor leg position. A high firing rate is interpreted by the CNS as a large leg movement, whereas a
low firing rate is interpreted as a small leg movement. In fact, the vast majority of sensory receptors
in animals use a change in firing rate to encode information about stimulus intensity.
Many sensory receptors can also display a phenomenon called sensory receptor adaptation.
If a stimulus is prolonged in duration, the sensory receptor firing rate will decline over time. Sensory
receptors generally differ in the speed of sensory receptor adaptation.

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Biol 224: Recording Nerve Action Potentials 33

Sensory receptor adaptation can be shown in a human volunteer. Have the volunteer close their
eyes and sit quietly with both hands on the lab bench. Drop a small piece of paper on the back
of the volunteer’s hand. The sensation associated with the paper initially hitting the hand
should be quite noticeable but will quickly fade, perhaps to even being undetectable. Several
types of stretch receptors are found in human skin; Pacinian corpuscles in particular display
very rapid sensory receptor adaptation.

Equipment Required
PC Computer
iWorx 214 data acquisition unit
USB cable
iWorx power supply
C-AAMI-504 input cable
C-ISO-N3 lead wires with needle electrodes (3)
Balsa wood and table clamp
Acetate copy of angles
Pasteur pipette (glass probe)
Pins

Experiment 1:
Equipment & Recording Setup
1.) Turn on iWorx 214 and open the LabScribe program. No device found? Tools, find
hardware.
2.) In the main window, pull down the Settings menu and select the Biology 224 folder. No
settings folder found? Refer to Lab 1, page 15.
3.) Open the settings file entitled Cockroach.
4.) Locate the C-AAMI-504 recording cable (Figure 1).

Figure 1: The C-AAMI-504 recording cable Figure 2: C-ISO-N3 lead wires with needle electrodes.

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Biol 224: Recording Nerve Action Potentials 34

5.) Plug the black AAMI connector on the end of the recording cable into the isolated Inputs
of Channels 1 and 2 of the iWorx 214
6.) Locate the C-ISO-N3 lead wires with needle electrodes (Figure 2).
7.) Attach the red, black, and green C-ISO-N3 lead wires to the corresponding sockets on
the lead pedestal of the C-AAMI- 504 recording cable. DO NOT INSERT THE LEADS
WIRES DIRECTLY INTO THE iWORX 214 BOX.

Electrical Noise
Electrical noise is the most common problem associated with the recording of bioelectric
signals. It radiates through the air and comes from electrical devices in the lab room or building such
as lights, power outlets, computers, monitors, and the power supplies. There are two major sources
of electrical noise: pickup and ground loops. Pickup is caused by electrical radiation that produces
currents in the electrodes and wires leading to the amplifiers in the recording system. The major way
to reduce pickup is the use of a Faraday Cage. A Faraday cage is a grounded, screened enclosure,
around the preparation and the electrodes. The enclosure separates the source of the radiation from
the electrodes.
Ground loops are a troublesome source of electrical noise caused by the ground cable itself
serving as an antenna for the noise radiating in the room. Using a Faraday cage to shield the
preparation and the recording electrodes does not remove the electrical noise caused by ground
loops. To avoid ground loops, you will use simple cables equipped with alligator clips, to connect
each device directly to the common grounding point on the iWorx 214 box. The C-AAMI-504
recording cable has a ground cable attachment, which will be placed through the coxa of the
cockroach leg and into the balsa wood.
To further improve recordings of the action potential a digital filtering function is applied to
the data as will be seen in the bottom channel of the iWorx recording.

Preparation of the Cockroach Leg


The hind legs of the cockroach are called the metathoracic legs, and they function in moving
the cockroach forward. The coxa is the upper portion of the leg, which attaches the leg to the thorax.
The trochanter acts like a knee and lets the cockroach bend its leg. The femur and tibia resemble
thigh and shin bones. The segmented tarsus acts like an ankle and foot.

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Biol 224: Recording Nerve Action Potentials 35

Coxa
Femur

Trochanter
Femur / Tibia Joint
This is the leg
manipulation point.
Neutral Position is indicated by
Tibia the line.
Notice the
spines

Tarsus

Flexion Extension

Figure 3. Cockroach metathoracic leg

1.) You will be given a metathoracic leg from a cold-anesthetized cockroach (Figure 3).
2.) Position the piece of acetate transparency illustrating angles over the balsa wood.
3.) Clamp the balsa wood and acetate transparency onto the bench top
4.) Place the cockroach leg over the acetate transparency so that the leg is positioned in the
neutral position (Figure 4) with the Femur-Tibia joint on the point of angles.
5.) The needles on the C-ISO-N3 lead wires with needle electrodes will serve as the
recording electrodes. Be careful when handling the needle electrodes. Handle the
needles themselves rather than the electrode base, as the needles are not firmly
anchored in the electrodes.
It is critical that you position the electrodes correctly the first time. The preparation will not work
if you pierce the leg more than once for each electrode.
6.) Place the needle electrode on the green ground (C) lead wire through the coxa and into
the balsa wood (Figure 4).

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Biol 224: Recording Nerve Action Potentials 36

7.) Place the needle electrode on the red (+1) lead wire near the end of the femur closest to
the tibia. This needle is the distal recording electrode for the preparation. Place the
needle electrode on the black (-1) lead wire at the proximal end of the femur. This
needle will function as the proximal recording electrode (Figure 4).

Figure 4: Cockroach leg drawing illustrating the correct position of the leg on the acetate
transparency. Arrows point to electrode placement locations and dashed lines indicate the angles
used in leg manipulations.

8). Place the Faraday cage over the entire set-up and make sure that the cage is connected
to a grounding point on the iWorx 214. Experiments may also be conducted with the
main room lights turned off to reduce electrical noise (see above).

Warning: The cockroach preparation used in this experiment is functional for a limited period of
time. To conserve time, complete all the exercises in each experiment before analyzing the data.

Experiment 1: Flexion and Extension of Chordotonal Organs


This experiment will explore the basic characteristics of the chordotonal organs, their
response to direction and intensity of leg movement. When the cockroach leg moves, flexion and
extension of the tibia cause changes in the length of the chordotonal organs. The chordotonal organs
span joints and have one or both ends attached to the cuticle. Flexion is the bending of a limb at a
joint, and will bring the tibia closer to the femur. Extension is the opposite of flexion, and moves the
tibia farther away from the femur. Refer to Figure 4 for help in determining which movement is
extension and which is flexion.

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Biol 224: Recording Nerve Action Potentials 37

Note: Action potentials from a given neuron will all be of the same amplitude, regardless of the
recording technique. Intracellular recordings from the same neuron can be seen in Figure 36.10 of
Russell et al. (2013) or Figure 44.10 of Russell et al. (2015). Action potentials recorded extracellularly,
as in this experiment, may vary greatly in amplitude. This is because an extracellular action potential
recording is a recording of the voltage changes resulting from the current that travels past the
recording electrodes on the outside of the nerve. The recorded action potential amplitude will vary
depending on the diameter of the neuron and the distance of the electrodes from the neuron. For
example, a large diameter axon will produce a larger amplitude action potential than a small
diameter axon because the large diameter axon has a greater surface area than the small diameter
axon. More current will leak across the greater surface area and this will result in a recorded action
potential of greater amplitude. Recall from the background information that many axons are present
within the cockroach leg. Therefore, if the recording electrodes are closer to one axon than another,
the current recorded by an action potential in the closer neuron will be greater than that in the other
axon because current dissipates over distance. These differences in action potential amplitude, allows
us to discriminate different neurons from a relatively simple extracellular technique. Action potential
amplitude variability can be observed below.

Notice the 3 different sized action potentials

Figure 5a (top) and 5b (bottom). LabScribe printout showing action potentials recorded from a cockroach
mechanoreceptor. Action potentials may be of similar amplitude (5a) or may show a large variation in
amplitude if the electrodes are recording from more than one axon (5b).

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Biol 224: Recording Nerve Action Potentials 38

Experimental Procedures
1.) Click the Record button. DO NOT CLICK STOP because LabScribe will automatically record
12 sweeps of data (each sweep is 500 ms long) and then the recording will stop. Click
Autoscale button or on the plus sign within the magnifying glass to scale your data
appropriately.
2.) The action potentials will have an amplitude greater than the baseline (Figure 5), but do
not be surprised if you do not see any activity without stimulation.
3.) Once the 12 sweeps of data have been recorded, right click on the initial sweep tab at the
bottom of the screen and rename the sweep Neutral.
4.) Press Record and quickly flex the tibia using the Pasteur pipette or pin to 40 degrees
and hold the leg in position for 5 seconds while the data is being recorded.
5.) Let the tibia return to its neutral position for 60 seconds.
6.) Once the program has completed recording the 12 sweeps of data right click on the
sweep of data that corresponds to the tibia being flexed (should be sweep 13) and
rename the sweep Flexion.
7.) Press Record and quickly extend the tibia using the Pasteur pipette to 40 degrees and
hold the leg in position for 5 seconds while the data is being recorded.
8.) Return the tibia to the neutral position.
9.) Once the 12 sweeps of data have been recorded, right click on the sweep of data that
corresponds to the tibia being extended and rename that sweep Extension.
*Tibial movement should result in a number of action potentials being generated.
10.) Select Save As in the File menu, type a name for the file. Save the file on your V: drive
and designate the file type as*.iwxdata.
Do not attempt to delete sweeps of data, iWorx automatically renumbers the sweeps if some
are deleted which makes it difficult to keep track of your data.

Before you can proceed to Experiment 2, you must determine whether your cockroach leg
responded more to flexion or extension. To establish this, scroll through the first few
sweeps of your flexion and extension data to see which type of movement resulted in more
action potentials being produced. Perform this task quickly as the leg could stop
functioning.

Data Analysis and Presentation


Proceed to Experiment 2 before you begin your data analysis for Experiment 1.
1.) Select the sweep of data you labeled Flexion.
2.) Scroll to the sweep of data where the greatest response to movement occurred (where
the largest number of action potentials are observed).
3.) Determine the firing rate of the mechanoreceptor, which is number of action potentials

Copyright 2019- Tracy Marchant & Sheri Fisher


Biol 224: Recording Nerve Action Potentials 39

per second. To calculate firing rate, count the number of action potentials on the
screen and divide that by recording time in seconds. Remember that each sweep of
data is 500ms in duration.
4.) If you have too many action potentials on the screen then you may take a subsample of
the recording to calculate firing rate. Using Double Cursors mode, position the cursors
on either side of the channel data until the T2-T1 located under the Record button is
200ms. Count the number of action potentials between the cursors and determine
the firing rate.
5.) Open Microsoft Word and paste a copy of Flexion data from Experiment 1 into a new
document. You can right click on the channel data that you want and select copy.
6.) Select the sweep of data you labeled Extension.
7.) Repeat steps 2 - 5. Write a single figure legend and conclusion for both Flexion and
Extension data. Show your calculation of Firing Rate for both types of movement.
You will also need to include axis labels.

Experiment 2: Phasic or Tonic Response and Adaptation


During this exercise, you will determine the effect that constant stimulation has on firing rate.
A reduction in neural response with continued stimulation is called sensory receptor adaptation. The
pattern of neural response reduction allows us to classify the sensory receptor as either phasic or
tonic. If the firing rate ceases or is greatly reduced when movement of the receptor stops, even
though a new position is maintained, such a response is considered to be phasic. Phasic, or rapidly
adapting, responses are typically associated with changes in the intensity of a stimulus. If action
potentials continue for as long as the stimulus is maintained and slowly decline overtime, the
response is termed tonic. Tonic, or slowly adapting, responses are typically associated with a
prolonged stimulus. Lastly, a response may be termed phasictonic. If the initial response to a
stimulus is a burst of action potentials, which is immediately followed by a large drop in firing rate
that settles into a tonic pattern as the stimulus is maintained, the response is phasictonic.

For this experiment the leg manipulator and computer person will have to work together
1.) In the previous experiment you determined whether your cockroach leg responded more
strongly to flexion or extension. In this experiment, move the leg in the direction that
gave the strongest response as determined by Experiment 1.
2.) Open a new LabScribe file by selecting File and New.
3.) Ensure that your cockroach leg is in the neutral position and Record a baseline set of 12
sweeps. Rename sweep 1 Neutral.
4.) Press Record and immediately position the tibia using the Pasteur pipette so that it is held
at the 20 degree (flexion or extension as determined by Exercise 1) position for the
recording of 12 sweeps.
5.) Return the tibia to the neutral position and allow the leg to rest for 60 seconds.

Copyright 2019- Tracy Marchant & Sheri Fisher


Biol 224: Recording Nerve Action Potentials 40

6.) Press Record and immediately position the tibia using the Pasteur pipette so that it is
held at the 40 degree position for the recording of 12 sweeps.
7.) Return the tibia to the neutral position and allow the leg to rest for 60 seconds.
8.) Press Record and immediately position the tibia using the Pasteur pipette so that it is
held at the 60 degree position for the recording of 12 sweeps.
9.) Rename the sweeps of data where the leg was initially moved Neutral to 20 Degrees,
Neutral to 40 Degrees and Neutral to 60 Degrees.
10) Select Save As in the File menu, type a name for the file. Save the file on your V: drive
and designate the file type as*.iwxdata.

Data Analysis and Presentation


1.) You want to create a figure that compares the change in firing rate over time for
movements to 4 leg angles.
2.) Determine 4 relative time periods (beginning, early middle, late middle and end) for each
of the four leg positions and the firing rates associated with those time periods.
3.) Given that each sweep is 500ms, Sweep 1 may be your Time = 0.5 seconds, while
Sweep 4 may be Time = 2 seconds, Sweep 7 = 3.5 seconds and Sweep 10 = 5
seconds.
You have 6 seconds of total recording time so there is some room to obtain data if,
for example, Sweep 1 data is too messy to differentiate between different action
potentials (which is common since you may have been moving the leg when the
recording started).
4.) Select the four sweeps of data, as determined in step 3, in your Neutral data and
determine the firing rates.
5.) Select the same four sweeps of data in your Neutral to 20 degrees data and determine
the firing rates.
6.) Select the same four sweeps of data in your Neutral to 40 degrees data and determine
the firing rates.
7.) Select the same four sweeps of data in your Neutral to 60 degrees data and determine
the firing rates.
8.) Remember that if the recording is unclear due to leg manipulation noise you may choose
the next sweep of data where you observe a large response to movement. Also, if
you have too many action potentials on the screen then you may take a subsample
of the recording to calculate firing rate.
9.) Construct an excel XY Scattergraph with straight lines and markers illustrating firing rate
over time for each leg position. Write a proper figure legend and conclusion for the
graph, focusing on differences within each movement type and on any differences
between them. Also indicate whether the observed responses were phasic, tonic, or
phasictonic.

Copyright 2019- Tracy Marchant & Sheri Fisher


Biol 224: Recording Nerve Action Potentials 41

Making this Excel graph can be tricky. These instructions will help you.
Type “Time” in the first cell (A1); “Neutral” in cell to the right (cell B1), “20º” in cell C1, “40º” and
“60º”. Enter the time in cells A2-A5, and the firing rates under the appropriate leg position and
beside the appropriate time. Select all the data in the spreadsheet and create an XY scatter graph
with lines. Once your graph is created you will notice there are only 3 lines of data while we need 4.
To fix this, right click on the graph, select data and switch rows/columns, click OK.

Group Assignment Due Today:


1.) A LabScribe printout illustrating the Cockroach leg stretch receptor response to Flexion and
Extension. Remember to label all axes. Show your calculations of Firing Rate.
2.) An Excel XY Scattergraph of the Cockroach leg stretch receptor firing rate at different leg
positions.
Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to include
your group number, lab day, all names (first and last) and Student ID# from your lab group members
on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Question to ponder: Refer to the definition of sensory receptor adaptation. What is the
significance of sensory receptor adaptation in the overall physiology and life of an animal?
Alternatively, what would life be like for an animal if there were not sensory receptor
adaptation?

Readings from the Scientific Literature: (Through the link below or on reserve in the Natural Sciences
Library)
Collin, S.P. 1985. The central morphology of mechanoreceptor afferent in the metathoracic leg of
the cockroach, Periplaneta americana (Insecta). Journal of Neurobiology. 16: 269-282.
French, A. S. and P.H. Torkkeli. 1994. The basis of rapid adaptation in mechanoreceptors. News in
Physiological Sciences 9: 158-161.
Kutchai, H. C. 2006. Generation and Conduction of Action Potentials. In Berne & Levy Principles of
Physiology (eds. M.N. Levy, B.M. Koeppen, B.A. Stanton). pp.31-41, Philadelphia, PA.
Elsevier Mosby.
Ridgel, A.L., S.F. Frazier, R.A. DiCaprio and S.N. Zill. 2000. Encoding of forces by cockroach tibial
campaniform sensilla: Implications in dynamic control of posture and locomotion. Journal of
Comparative Physiology A. 186:359-374. Click here for download
Sperelakis, N. 1996. Cable Properties and Propagation Mechanisms. In Essentials of Physiology
(eds. N. Sperelakis, R.O. Banks). pp.51-59, Boston, MA. Little, Brown and Company.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Skeletal Muscle Physiology 42

LAB EXERCISE #4: SKELETAL MUSCLE


PHYSIOLOGY
Recommended Textbook Reading:
Sections 45.2d, 45.9 and Chapter 46 of Russell et al (2016) Canadian edition
Section 45.3d and Chapter 46 of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION
Skeletal muscle is an excitable tissue in that an action potential (AP) is generated along the
plasma membrane of muscle cells. The generation of a skeletal muscle AP is controlled by motor
neurons originating from the central nervous system (CNS) of animals. Motor neurons release a
neurotransmitter (acetylcholine) at a specialized synapse called a neuromuscular junction (or motor
endplate). Release of acetylcholine causes a muscle AP that will stimulate contraction of the muscle
cell. In vertebrates, motor neurons always excite muscle fibers (i.e. cause them to contract), however
for invertebrates, motor neurons can be both excitatory or inhibitory depending on the
neurotransmitter released and receptor it binds to. Skeletal muscle contraction is generally under
“voluntary” control by the CNS although “involuntary” reflex arcs have very important roles to play
in terms of coordinating overall body position, posture, and locomotory patterns.
A unique feature of tetrapod vertebrates is the presence of limbs designed for movement in a
terrestrial environment. Movement of limbs is generally accomplished by coordinating the
contraction and relaxation of antagonistic muscle groups. Tendons are used to attach muscles to
bones and the coordinated contraction and relaxation of antagonistic muscle groups are responsible
for changes in limb position. Information from a variety of sensory receptors (e.g. stretch receptors)
is sent to the CNS and assists in the coordination of muscle contraction and overall limb movements.
Understanding the role of sensory information and reflex arcs in walking and other locomotory tasks
in humans is a very active research area today. This knowledge has important clinical implications in
the development of effective rehabilitation treatments for muscle disease, brain and spinal cord
injuries, and in sports medicine.
Electrical activity associated with skeletal muscle APs can be recorded from the surface of the
body. The recording of muscle activity is called an electromyogram (EMG) and is accomplished by
placing recording electrodes on the skin overlying the muscle group under study. EMGs are a non-
invasive way to assess muscle function. These recordings are then used to provide important
information about the regulation and control of limb movement in humans and other animals.
This lab exercise will examine EMGs in a human volunteer. Two experiments will be conducted
to assess muscle activity under different situations.
Equipment and Materials Required
PC Computer and iWorx 214 data acquisition unit
PRH-100 Percussion hammer
C-AAMI-504 ECG/EMG cable and electrode lead wires
Disposable snap electrodes
FT-325 Hand Dynamometer

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Skeletal Muscle Physiology 43

Cotton swabs and rubbing alcohol


Calibration weight
Tape measure

Important Notes: Volunteers for Experiment 1 need to wear shorts. Please bring these with you to this
lab exercise. A changing room is available.

EXPERIMENT 1: EMG RECORDING DURING ACHILLES REFLEX


Skeletal muscles contain specialized receptors which convey information about muscle
length, tension, and pressure to the central nervous system. The mechanosensory receptors
responsible for providing information about the length, or the rate of change of the length, of a
muscle are called muscle spindles. Muscle spindles are arranged in parallel with muscle fibers and
are stretched when an external force, such as a tendon tap, stretches the muscle. The stretching of
a muscle causes excitation of its muscle spindles and results in a reflex contraction of the muscle.
The reflex response is known as a stretch (myotatic) reflex.

Stretch reflexes involve large diameter sensory axons and contain a small number of synapses
in the circuit; factors that result in a minimal time delay from when the muscle is stretched and when
the muscle contracts. Stretch reflexes have a monosynaptic pathway because the sensory afferent
nerves from the muscle spindles enter the spinal cord and synapse directly with motor neurons,
rather than with interneurons. When the motor neurons are stimulated they trigger the contraction
of the same muscle group. The same sensory neuron also synapses to an interneuron that connects
to another motor neuron, which stimulates the antagonistic muscle group, completing the reflex arc.

Here you will record EMG activity in the calf muscle group responsible for extension of the foot
during the Achilles reflex. The calf muscle group is composed of the gastrocnemius (which consists
of two branches or heads) and the soleus (which lies underneath the gastrocnemius). The
gastrocnemius and soleus merge at the base of the calf and are joined to the Achilles tendon by
tough connective tissue. Recording electrodes will be placed over the calf muscle, and an EMG will
be recorded while the Achilles tendon is struck gently with a percussion hammer. The EMG will
primarily reflect muscle activity from the gastrocnemius. The PT-104 pulse plethysmograph will be
attached to the head of the percussion hammer to record the exact time when the tendon is struck.

Have the volunteer from your lab group change into shorts and remove their shoes. For this
experiment, the volunteer should sit comfortably on the lab bench so that their thighs are supported
by the lab bench but their feet are dangling free and can swing under the bench. The volunteer
should flex their foot so that it is at a 90° angle. The volunteer should sit as quietly as possible to
minimize noise from extraneous muscle activity in the EMG. Another group member will be
responsible for placing the recording electrodes and initiating the Achilles reflex. The Achilles tendon
is located above the heel and connects the gastrocnemius muscle to the tarsal bone of the foot. Tap
the tendon with the wide end of the reflex hammer a few times to locate a point on the tendon,
which produces a consistent contraction of the calf muscle and a downward movement of the foot
(plantar flexion). The opposite, upward movement is known as dorsiflexion. The third group
member will be responsible for operating the computer and recording the data.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Skeletal Muscle Physiology 44

Equipment & Recording Setup


1.) Turn on the iWorx 214 and open the LabScribe program. No device found? Tools, find
hardware
2.) On the main window, pull down the Settings menu open the iWorx settings file called
Achilles. See Lab 1, page 15 if the settings folder is not present.
3.) Plug the PRH-100 percussion hammer into the Non-Isolated Input Channel 2 of the
iWorx214 (Figure 1). Try a few gentle taps of the percussion hammer to ensure that
hammer strikes are being recorded in the Tendon Tap channel of LabScribe.
4.) Insert the black AAMI connector on the end of the EMG cable into the Isolated Inputs of
Channels 1 and 2 of the iWorx214 (Figure 1).
5.) Use a cotton swab soaked with rubbing alcohol to clean and gently abrade three regions
on the calf of the left leg. One area is near the ankle. The second is in the middle of the
calf muscle and the third area is about 3 inches below the back of the knee. Let the areas
dry. PUT THE LID BACK ONTO THE TOP OF THE RUBBING ALCOHOL BOTTLE.
6.) Remove the plastic disk from a disposable electrode and apply it to one of the abraded
areas. Repeat for the other two areas.
7.) Attach three color-coded electrode leads to the ground and Channel 1 inputs on the lead
pedestal and snap the other ends onto the disposable electrodes (Figure 1), so that:
• the red (+1) lead wire is attached to the electrode near the back of the knee.
• the black (-1) lead wire is attached to the middle of the calf muscle. The green (C)
lead wire is attached to the electrode on the ankle to function as the ground.

Hint: If the EMG recording is noisy, abrade the skin areas again with alcohol or reposition the electrodes
until a fairly clear signal is recorded. Ask the volunteer to minimize other leg movements during the
experiment.

Red

Black

Green

Figure 1. Cables and connections to the iWorx 214 used for recording a human
electromyogram (left panel). The positions of the various electrodes on the calf
muscle are also illustrated (right panel).

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BIOL 224: Skeletal Muscle Physiology 45

8.) Click Record and then instruct the subject to point his or her foot down and up to
demonstrate the type of EMG that occurs during calf contraction and relaxation.
Click AutoScale on the EMG Calf channel. Click Stop to halt the recording.

Figure 2: Drawing illustrating the location of the Achilles


tendon.

Image from Wikemedia.org. Reproduced under creative


commons.
Original images by: By Montrealais at en.wikipedia [Public
domain], from Wikimedia Commons

Experimental Procedures
1.) Type Achilles Tendon Reflex in the Mark box that is to the right of the Mark button.
2.) Click Record. Press the Enter key on the keyboard to mark the recording.
3.) Instruct the subject to close their eyes and flex their foot so that it is at a 90° angle to the
leg.
4.) Tap the subject’s Achilles tendon to elicit the stretch reflex. You should obtain a recording
similar to that shown in Figure 3. (Click AutoScale)
5.) Record a total of ten trials using the same tapping force that result in measurable recordings.
6.) After the tenth trial, click Stop to halt recording. Select Save in the File menu. Do not save
to the R drive as the system will crash and all data will be lost.

Figure 3. LabScribe window


showing typical data from an
electromyogram (EMG) obtained
from a human calf muscle during
the Achilles tendon reflex. The
top panel is the EMG, whereas
the lower panel is a recording of
the percussion hammer as it
struck the Achilles tendon.

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BIOL 224: Skeletal Muscle Physiology 46

Data Analysis and Presentation:


1.) Scroll to the beginning of the data so that the first trial is displayed on the main window.
2.) Use the Display Time icons to adjust the Display Time of the Main window to show the
signal made by tapping the tendon and the EMG response on the main window. This
trial can also be selected by placing the cursors on either side of the tendon tap and
the subject’s EMG response and clicking the Zoom between Cursors button on the
LabScribe toolbar to expand the complete trial to the width of the main window.
3.) Click the Analysis Window icon in the toolbar or select Analysis from the Windows
menu to transfer the data displayed in the main window to the analysis window.
4.) Look at the Function Table that is above the display of the EMG Calf channel displayed in
the Analysis window. The mathematical function, T2-T1, should appear in this table.
The value for T2-T1 is seen in the table across the top of the EMG Calf channel.
5.) Use the mouse to click on and drag a cursor to the onset of the signal recorded from the
hammer in the Tendon Tap channel. Drag the other cursor to the peak of the EMG
wave in the EMG Calf channel.
6.) Once the cursors are placed in the correct positions for determining the reflex conduction
time, record the value for T2-T1 in the Journal. The value can be recorded in the
online notebook of LabScribe by typing its name and value directly into the Journal.
The functions in the channel pull-down menus of the analysis window can also be
used to enter the name and value for T2-T1 into the Journal. To use these
functions:
• Place the cursors at the locations used to measure the reaction time.
• Transfer the name of the T2-T1 function to the Journal using the Add Title
to Journal function in the EMG Calf Channel pull-down menu.
• Transfer the value for T2-T1 to the Journal using the Add Ch. Data to
Journal function in the EMG Calf Channel pull-down menu.
7.) With the Analysis window open, use the bottom scroll bar to move the data from the
second trial onto the window. If needed, use the Display Time icons to adjust the
width of the analysis window to show both the signal from the tendon tap and the
subject’s EMG response on the same window.
8.) Repeat these steps to measure the reflex conduction times from the other nine trials.
9.) Once the times for all ten trials have been measured and recorded, use the values from
the Journal to determine the mean reflex conduction time of the subject. Discard the
longest and shortest times from the data set, and determine the average of the eight
trials. Click Save. Hint: you can copy and paste the reflex times into Excel and use the
average function to quickly find the mean reflex conduction time.
10.) Measure the distance between the belly of the calf muscle and the location of the
sensory-motor synapse in the spinal cord. For the purpose of this exercise,
assume that the sensory-motor synapse is at spinal segments L5 and S1, which are
just above the top of the hipbone. Note: Since the signal travels to and from this

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BIOL 224: Skeletal Muscle Physiology 47

location, you must multiply the measurement by 2 to determine the total length of the
nerve path.
11.) Even though this stretch reflex is known as a monosynaptic reflex, the pathway also
includes the neuromuscular synapse. Assume that synaptic transmission takes
about 0.5 msec, calculate the conduction velocity in the nerves composing this reflex
pathway by the equation:

Conduction Velocity Total Path Length (meters)


=
(meters/sec)
Mean Reflex Time (sec) – Synaptic Transmission Time (sec)

12.) Open the Microsoft Word program and, paste a copy of a typical EMG and tendon tap
from Experiment 1 into a new document. Write a figure legend. Include the
calculated value of the conduction velocity of the action potentials in this reflex
arc.

EXPERIMENT 2: EMG RECORDING DURING DIFFERENT GRIP STRENGTHS


The strength of a striated muscle contraction depends on the amount of electrical activity in
the muscle. To quantify the amount of electrical activity in a muscle, EMG data is mathematically
transformed. One of the most common transformations used is the integration of the absolute
values of the amplitudes of the EMG spikes. Through this transformation, the area under the graph
of the absolute integral of the EMG is linearly proportional to the strength of the muscle contraction.
In this experiment, a hand dynamometer (Figure 3) will be used to measure a volunteer’s grip
strength as the EMG activity from the forearm muscles are recorded. The EMG activity will be related
to the grip strength by plotting the maximum grip strength as a function of the area under the
absolute integral of the EMG activity during the muscle contraction.
Vertebrate skeletal muscle is made up of groups of muscle fibers that are innervated by
motor neurons. A single motor neuron has a branched axon, allowing it to innervate multiple muscle
fibers (from a few up to several thousand). The motor neuron and the muscle fibers it innervates are
called a motor unit. When the motor neuron undergoes an action potential, all of the associated
muscle fibers are stimulated and contract. By increasing the number of active motor units the
strength or force of the muscle contraction will increase in a principle known as motor unit
recruitment. Since there is a finite number of motor units in each muscle, once all of the motor units
have been recruited no additional force can be generated. At this point we will observe a plateau of
muscle force as we reach the maximal level of muscle activity.

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BIOL 224: Skeletal Muscle Physiology 48

Red

Black

Green

Figure 3. The FT-325 hand dynamometer and C-AMMI cable with electrode lead wire
used for recording a human forearm electromyogram during Experiment 2 (left panel).
The positions of the various electrodes on the forearm muscle are also illustrated (right
panel).
Equipment & Recording Setup
1.) Open a new main window in the LabScribe program.
2.) On the main window, pull down the Settings menu and select the Biology 224 folder.
3.) Open the iWorx settings file called Grip Strength.
4.) Plug the DIN8 connector to the FT-325 hand dynamometer into the Non-Isolated Input
Channel 3 of the iWorx214 and leave the black CAAMI connector on the end of the EMG
cable connected to the Isolated Inputs of Ch 1 and 2 of the iWorx214 (Figure 4).
5.) Select another volunteer from your group and have them sit comfortably on a lab chair.
Remove all jewelry from their wrist and role up their sleeve. During the experiment, the
volunteer should rest their forearm on the lab bench, palms facing up. This will minimize
noise from other muscles as the EMG is recorded.
6.) Use a cotton swab soaked with rubbing alcohol to clean and gently abrade three regions on
the medial (inner) side of their dominant arm for electrode attachment (Figure 4). One
area is near the wrist, the second is in the middle of the forearm, and the third area is
about 2 inches from the elbow. Let the areas dry.
7.) Remove the plastic disk from a disposable electrode and apply the electrode to one of the
abraded areas. Repeat for the other two areas.
8.) Attach three color-coded electrode leads to the ground and Channel 1 inputs on the lead
pedestal and snap the other ends onto the electrodes (Figure 3), so that:
• the black (-1) lead wire is attached to the electrode in the middle of the forearm.
• the red (+1) lead wire is attached to the electrode near the elbow
• the green (C) lead wire attached to the electrode on the wrist to function as the
ground.

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BIOL 224: Skeletal Muscle Physiology 49

9.) Click Record and then instruct the subject to squeeze the dynamometer bulb to ensure that
EMG and dynamometer data are being recorded in the upper and lower channel
windows, respectively of LabScribe. Click Double Display Time and AutoScale on both
channels so you can observe entire squeezes. Click Stop to halt the recording.
Calibration of the Hand Dynamometer:
Physiological variables recorded by the iWorx 214 are initially displayed in LabScribe with
electrical units of millivolts (mV). In this experiment, the hand dynamometer is being used to
measure grip strength, which is technically a mechanical force. It is thus inappropriate to present
force data in this experiment with mV units. Instead, the Units Conversion feature of LabScribe will
be used to automatically convert the mV data to a unit more representative of a mechanical force,
such as kilogram (kg).

1.) Lay the hand dynamometer down on the bench top. Click the Record button on the
LabScribe Main window and record for ten seconds.
2.) Continue to record as you place the calibration weight on the bulb of the hand
dynamometer. Record for an additional ten seconds after the weight is placed on the
bulb. Click the Stop button.
3.) Click the AutoScale button on the bottom Muscle Force channel. Use the Double Display
Time icon to adjust the Display Time of the Main window and display the force
recording before and after the weight was placed on the hand dynamometer.
4.) Click on the Double Cursors button on the LabScribe toolbar. Place one cursor on the force
recording made before the weight was placed on the bulb. Place the other cursor on
the recording after the weight was placed on the bulb.
5.) Open the Channel Menu of the Muscle Force channel by clicking on the down arrow to the
left of the channel’s title:
6.) Select Units from this menu and Simple from the submenu to open the Simple Units
Conversion dialogue window (Figure 4).
7.) Put check marks in the boxes next to Apply Units to new data and Apply Units to all blocks.
8.) In the middle of the window is an array of four boxes. For each cursor, the value in the box
on the left is the voltage at the position of the cursor on the recording window.
9.) In the box on the right, enter the value of the unit that equals the voltage on the left:
• For Cursor 1, type zero (0) in the box on the right. This cursor is on the portion of the
recording when no weight was placed on top of the hand dynamometer.
• For Cursor 2, type the mass of the calibration weight in the box on the right.
• Type the name of the unit, kilogram or kg, in the Unit Name box. Click the OK
button.

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BIOL 224: Skeletal Muscle Physiology 50

Enter the
calibration
weight here

Figure 4. The LabScribe dialog box used to


convert electrical units to a more
appropriate unit for data analysis. Note that
the values displayed in the boxes as you
proceed through the calibration will differ
from those shown above.
Experimental Procedures:
1.) The volunteer should sit quietly with his or her dominant forearm resting on the table top,
palms facing up. Have the volunteer squeeze the hand dynamometer very hard for
a few seconds. When they have finished the initial squeeze, Autoscale the
channels. This is done to ensure the subsequent squeezes will be visible.
2.) The subject will squeeze his or her fist around the hand dynamometer seven times, each
contraction should be three seconds long followed by a period of at least
three seconds of relaxation (Figure 6). Each successive contraction should be
approximately two, three, four and five times stronger than the first contraction
with the fifth contraction being the maximal. Contractions six and seven should also
be the subjects’ maximum contraction strength.
Note: Your data should look similar to Figure 5. Try to apply a constant force for each
three-second squeeze for each grip strength. The Grip Strength data should display a ‘flat
top’ appearance.
3.) Type Increasing Grip Force in the Mark box to the right of the Mark button.
4.) Click the Record button to begin the recording; then, press the Enter key on the keyboard
to mark the beginning of the recording. After the recording is marked, tell the
subject to begin squeezing the hand dynamometer following the procedure
outlined in step 1. In the relaxation period after the last contraction, click the Stop
button.
5.) Click the Double Display Time and AutoScale buttons for Channel 1 Muscle Activity
(EMG), and Channel 3 Grip Strength (Muscle Force).
6.) Once your data looks similar to Figure 5, Save the data file.

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BIOL 224: Skeletal Muscle Physiology 51

Figure 5. Representative LabScribe recording obtained during an experiment measuring


forearm muscle (EMG) activity (top channel) and grip strength (lower channel) in a
human volunteer. Each grip was held for 3 seconds using a constant strength with a 3
second rest in between each successive grip.

Data Analysis and Presentation:


1.) Use the Display Time icons to adjust the Display Time of the Main window to show the
five progressive and two maximal muscle contractions on the Main window.
2.) Click on the Analysis icon in the LabScribe toolbar to use the analysis window functions.
3.) Look at the Function Table that is above the uppermost channel displayed in the
Analysis window. The mathematical function, Abs. Area should appear in this table.
The value for Abs. Area, on each channel are seen in the table across the top
margin of each channel.
4.) Using Double Cursors mode, drag the cursors to the beginning and end of the first
muscle contraction in order to measure the absolute areas under the first muscle
contraction and corresponding EMG activity.
5.) Use the channel pull-down menus of the Analysis window to automatically enter the
names and values of the absolute areas to the Journal.
Note: You cannot have a negative value for muscle activity. If you do, adjust the cursors
and re-measure.
6.) Save the data.
7.) Use the data in the Journal to draw a Microsoft Excel XY Scattergraph (straight lines with
markers) relating muscle activity (x-axis) to grip strength (y-axis). Be sure to include
an appropriate figure legend and conclusion.

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BIOL 224: Skeletal Muscle Physiology 52

Group Assignment Due Today:


1.) A LabScribe printout illustrating the results from Experiment 1. Show your conduction
velocity calculation.
2.) An XY Scattergraph illustrating the results from Experiment 2.

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to
include your group number, lab day, all names (first and last) and Student ID# from your
lab group members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Question to ponder: Recall the definitions of motor-unit and recruitment. Why is it


useful to divide only skeletal muscle into motor units? Why not the heart?

Readings From The Scientific Literature: Through the link below or on reserve in the natural sciences
library

Biro, A., L. Griffen and E. Cafarelli. 2007. Reflex gain of muscle spindle pathways during fatigue.
Experimental Brain Research 177: 157-166. Click here for download

Cope, T.C. and A.J. Sokoloff. 1999. Orderly recruitment among motoneurons supplying different
muscles. Journal of Physiology 93: 81-85. Click here for download

Guyton, A.C. and J.E. Hall. 2006. Contraction of Skeletal Muscle. In Textbook of Medical
Physiology (eds W. Schmitt & R. Gruliow). pp. 72-84. Philadelphia, PA. Elsevier Inc.

Levy, M.N., B.M. Koeppen, B.A. Stanton. 2006. Skeletal Muscle. In Berne and Levy Principles of
Physiology (eds W.R. Schmitt & A. Hall). pp165-177. Philadelphia, PA. Elsevier Inc.

Schwellnus, M.P. 2007. Muscle cramping in the marathon: aetiology and risk factors. Sports
Medicine 37: 364-367. Click here for download

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Osmoregulation 53

LAB EXERCISE #5: OSMOREGULATION


Recommended Textbook Reading:
Section 5.4 and sections 50 – 50.2 of Russell et al. (2016) Canadian edition
Section 4.4 and sections 42 - 42.3 of Russell et al. (2018) Canadian edition
Osmosis is the movement of water across a semipermeable membrane (biological or
artificial), from an area of low solute concentration (hypotonic or hypo-osmotic) to an area of high
solute concentration (hypertonic or hyperosmotic). If the concentration of solute and water become
equal on either side of the membrane, the solution is deemed to be isotonic or iso-osmotic. Osmosis
is a type of passive transport that requires that the membrane restrict the movement of at least one
solute but not water (i.e. be semipermeable). Osmoregulation is simply the regulation of water and
ion concentrations in the body. Many biological membranes, including the plasma membrane, are
semipermeable.
There are two types of solutes, penetrating solutes and non-penetrating solutes. Solutes
that can diffuse across the cell membrane are penetrating solutes, while those that cannot diffuse
across the cell membrane are non-penetrating solutes. Solutes such as inorganic ions and large
organic molecules are incompatible with the plasma membrane structure and do not easily cross the
plasma membrane. In contrast, water is a relatively small molecule possessing high kinetic energy
and it moves freely through the aquaporins embedded within the plasma membrane along any
solute gradient that might be present. The volume of water that moves during osmosis can be
substantial, and is directly related to the magnitude of the impermeable solute concentration
gradient across the plasma membrane. Sufficient water can move to cause the cell to shrink (crenate)
or swell, or to otherwise disrupt normal cell physiology.
The effects of osmosis are exceedingly important to animals, especially those from an aquatic
environment. These animals live in direct contact with an aqueous solution, and the membranes of
their body surfaces are semipermeable. Thus, whole body water and solute concentrations are
influenced directly by their environment. Many aquatic animals possess specific tissues and organs
designed to regulate body (extracellular) water and solute concentrations. Animals that maintain
body solute concentrations different from the environment are called osmoregulators. All
freshwater animals are osmoregulators and are generally hyperosmotic to their environment (low
environmental salt content). Thus, freshwater animals are faced with body weight gains and losses
depending on the osmotic gradient that exists in their environment. Some marine animals do not
maintain their osmotic concentrations different from the environment and are called
osmoconformers. Sodium (Na+) is the predominant cation in the extracellular fluid of multicellular
animals and in seawater. By osmoconforming, some animals take advantage of the high level of Na+
in seawater so that minimal energy and body structures are needed for osmoregulation. Generally,
the large volume of water in the ocean ensures minimal fluctuations of the osmotic environment
and osmoconformers are usually exposed to a relatively stable environment. However, marine
animals living in tidal pools within the intertidal region experience fluctuations in the osmotic
environment, either due to the evaporation of water from the pool or because of dilution due to rain
water. Animals trapped in a tidal pool must be able to adapt to short-term osmotic stress and survive

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BIOL 224: Osmoregulation 54

for about 12 hours until the next tide. Thus, many invertebrates living in the intertidal zone are able
to osmoregulate at least for a short length of time. Other intertidal invertebrates (e.g. barnacles) use
behavioural mechanisms (e.g. closing up their shells) to simply avoid the osmotic stress associated
with a tidal pool.
The exact type of osmoregulatory system depends on the animal group and is influenced by
the nature of the aquatic environment (i.e. freshwater versus marine). However, most
osmoregulatory systems involve the active transport of ions across an epithelial surface, with water
following the ion gradient by osmosis.
Terrestrial invertebrates evolved from freshwater or littoral ancestors and therefore, their
blood concentrations are between 250 and 400 mOsm (milliosmolar). In contrast, seawater has a
milliosmolar concentration of about 1000 mOsm. For land invertebrates there two problems
associated with osmoregulation. First, salts have to be consumed, as they are not readily available
on land. Second, water must be obtained and preserved within the animal, since evaporation from
the body will always occur. The external skin of terrestrial invertebrates functions to let oxygen in
and carbon dioxide out as well as preventing water and ion loss. The internal tissues of annelids are
hyperosmotic relative to the freshwater soil surroundings. As a result, annelids take in water
osmotically and produce hyposmotic urine to get rid of excess water. In annelids, excretion and
osmoregulation are accomplished by a highly developed organ system called metanephridia
Equipment And Materials Required
PC Computer
Sheep red blood cells and plasma
Microscope slides, coverslips, compound light microscope and ocular micrometer
Pasteur pipettes and bulbs, test tubes
NaCl solutions (unknowns A and B) in 0.05 M phosphate buffer (pH = 7.4)
Seawater (Instant Ocean) – diluted to various concentrations
Ultrapure water
Freshwater (dechlorinated tap water)
Plastic beakers
Containers with earthworms
Electronic Balances
Tape
EXPERIMENT 1: OSMOREGULATION IN ANNELID WORMS
This experiment will measure osmotic water movement to and from the body of living
invertebrate animals exposed to various water treatments. Each group will be assigned five
earthworms to test in this experiment. Handle the animals carefully and gently.
There is a 30-minute incubation period in this experiment. You should start Experiment 1 and
then complete Experiment 2 during this incubation period.

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BIOL 224: Osmoregulation 55

Collecting Experimental Data

Safety Issue: You will be removing solution from large carboys. The carboy hoses are double
clamped to prevent leakage of solutions. When you have obtained your solutions, make sure
the hoses are double clamped. If you do not, be prepared to mop up any spills.
1.) Take five plastic beakers to the area where the solutions are kept. Pour about 50mL of the 5%,
20%, 40%, and 60% concentrations of seawater, and the unknown % seawater concentration
into the plastic beakers, make sure you have appropriately labeled the beakers. Bring these
to your lab bench. Be careful not to spill any liquid on your keyboard, the iWorx or other
computer equipment.
2.) Take another small plastic cup to the container, gently pick up five worms, rinse them in the basin
of water and place them in your cup and return to your lab bench.
3.) Gently blot the worms on a paper towel to remove excess water from its body surface.
4.) Electronic balances, similar to the one pictured in Figure 1, will be located through the lab room.
Proceed to the balance closest to your lab bench, with paper and pen, a weighing dish, your
worms and beakers of solution.

Figure 1: ScoutPro electronic


balance by OHaus.

5.) Place the weighing dish on the balance platform and press zero (or tarre). When the balance reads
0.00g, place a worm into the weighing dish. Record the weight and place the worm into a
seawater dilution. *Make sure the unit of measurement is Grams. Ask for assistance from a
lab demonstrator in the even that the balance has been changed to Newtons.
It is IMPERATIVE that you keep track of the weight of the earthworm and what treatment you
place it in.
6.) Repeat this procedure for the remaining earthworms.
7.) Keep the animals in the beakers for 30 minutes. Then, reweigh the worms using the
procedures described in steps 4 and 5 above.
8.) Return the animals to the appropriate container after the second weighing. Do not leave the
worms in water once you have completed the second weighing.

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BIOL 224: Osmoregulation 56

9.) *Clean up: Thoroughly rinse and dry the plastic beakers with paper towel and return them to your
bench.
Bring data of initial and final weights and associated seawater dilution for each worm to your lab
demonstrator.
Data Analysis And Presentation
1). Express the body mass of each animal at the end of the 30-minute incubation period as a
percentage of its initial mass (i.e. Final / Initial x 100). A value greater than 100% means that
the animal has gained water during the experiment, whereas a value less than 100% means
the animal lost body water.
2.) Your lab demonstrator will collect results from all groups and provide you with class data for
the various treatment conditions, including an unknown.
3.) Use the class data provided to you to create an Excel XY Scattergraph. Percent of seawater will be
on the X-axis and the percent of initial mass of the earthworms during the incubation period
will be on the Y-axis. There should be 4 data points on your graph. You CANNOT plot the data
for the unknown concentration of seawater.
4.) We now want you to add a trendline to the class data that will automatically choose the line of
best fit between your data points. The trendline function can be found in Excel under the
Layout tab in the Analysis grouping. Click on trendline and select More trendline options.
Click on the Linear Trendline and select Display Equation on Chart located at the bottom of
the page.
5.) The equation that appeared on your chart can be used to determine the concentration of the
unknown seawater. To do this, you will substitute the weight change of the earthworm (Y)
into the equation and calculate the concentration of the unknown seawater (i.e. solve for X).
6.) Include the calculation on your graph and write an appropriate figure legend and conclusion.

EXPERIMENT 2: OSMOTIC EFFECTS ON ANIMAL CELLS


When Sodium Chloride (NaCl) is dissolved in water, it dissociates into Na+ and Cl-; these
solutes are non-permeating solutes meaning they cannot diffuse through the cell membrane. In this
experiment, you will measure the diameter of sheep red blood cells (erythrocytes) exposed to NaCl
solutions of "unknown" concentration to determine the effect of osmotic concentration on water
movement across the cell membrane of animal. The cells will swell or shrink depending on whether
or not the NaCl solution is hypertonic or hypotonic relative to the intracellular fluid. By its very
nature, blood plasma (the non-cellular fluid of blood) is isotonic relative to the cytoplasm of red
blood cells. Comparison of the diameter of cells placed in the NaCl solutions with that measured in
cells exposed to plasma will allow you to determine whether the NaCl solutions are hypotonic or
hypertonic relative to the red blood cell cytoplasm.

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BIOL 224: Osmoregulation 57

Collecting Experimental Data


Safety Issue: In this experiment, you will be handling blood. You must wear the appropriate
personal protective equipment, when you conduct this experiment. Gloves are provided.
You may choose to wear a lab coat during this experiment as well, these will not be provided.
If you get any blood on the counter top, please notify your teaching assistant who will bring
a cleaning solution to your bench.
1.) Locate the two small test tubes containing unknown I or unknown II NaCl solution.
2.) Using a Pasteur pipette, add 3 drops of sheep erythrocytes to the unknown I test tube.
3.) Agitate the solution in the test tube gently, and let it incubate (sit at room temperature) for 10
minutes.
4.) During the incubation period of unknown I, prepare a slide of the isotonic control by placing one
drop of blood (erythrocytes kept in blood plasma) on a clean microscope slide and cover with
a cover slip. Be sure to label the slide. Proceed to measuring blood cells. Once cells have been
measured return to step 5.
5.) Add 3 drops of sheep erythrocytes to the unknown II test tube, gently agitate the solution in the
test tube and let it incubate for 10 minutes.
6.) During the 10-minute incubation period of unknown II place one drop of the erythrocyte /
unknown I NaCl mixture on a clean microscope slide and cover with a cover slip. Proceed to
measuring blood cells. Once cells have been measured return to step 7.
7.) Place one drop of the erythrocyte / unknown II NaCl mixture on a clean microscope slide and
cover with a cover slip. Proceed to measuring blood cells.
Measuring Blood Cells
1). You will be provided with a compound microscope fitted with an ocular micrometer. The
micrometer is essentially a small ruler viewed through the lens of the microscope. This allows
the diameter of objects on a slide to be measured.
2.) Place a slide on the microscope stage and focus on the erythrocytes with the 40x objective.
Remember to always begin by first focusing on the lowest power objective lens before you
move to high power lenses. DO NOT USE THE 100x LENS.
3.) With the micrometer, measure the diameter of a cell by counting the number of micrometer
divisions from one side of a cell to the other. Only measure cells lying flat on the slide.
4) Measure a total of 10 cells from the slide and record the micrometer divisions in an Excel
spreadsheet. The same individual should perform all of the measurements from all 3 slides to
reduce discrepancies.
5.) *Clean up: Blood contaminated slides, cover slips and Pasteur pipettes (not including the Pasteur
pipette bulbs) need to be placed into a bleach solution located in 2 sinks in the lab room.

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BIOL 224: Osmoregulation 58

Data Analysis and Presentation


1.) Calculate the average number of micrometer divisions for the plasma control (blood alone) and
each unknown NaCl solution.
2.) Provide your lab demonstrator with the average number of micrometer divisions; they will collect
results from all groups under her/his supervision and provide you with a class average.
3.) Convert the micrometer divisions to actual cell size (µm) using the following conversion factor for
the 40x objective: 40 dashes = 0.1mm (i.e. 100 µm). Simply set up a formula within Excel
similar to =A2*100/40 where A2 is the cell where you entered your micrometer divisions.
4.) You will be provided with data from at least 10 other groups. Enter the data provided into Excel
and calculate the class average. In Excel highlight the data of interest, click on the arrow
beside the sum symbol S, and select average.
*Statistical analyses were introduced in Lab 2. Information on standard deviation, confidence limits
and standard error of the mean can be found on page 25 of lab 2.
5.) Determine the standard deviation for each blood treatment group average. A few cells below the
average type =STDEVS. A cursor will be within the parentheses, to add data within the
parentheses highlight the same values you used for finding the average (do not include the
average when highlighting data). The standard deviation is being calculated so the confidence
intervals and standard error of the mean can be found.
6.) For each blood treatment group average find the 95% confidence interval. In an empty cell type
=confidence.norm. The cursor will be within parentheses. Three values must be entered within
the parentheses; alpha = 0.05, standard deviation is the value that was determined in step
12, and size is how many students (n) contributed to the class data.
7.) Calculate the upper and lower confidence limits for each cue type. To each blood treatment group
average, add the confidence interval to find the upper confidence limit and then subtract the
confidence interval to find the lower confidence limit.
8.) Find the standard error of the mean (SEM). There is not a built-in formula for SEM within excel.
The formula is SEM = (STDEVS / √n). To find SEM using Excel type = and click on the standard
deviation you determined earlier. After the standard deviation value enter the divide symbol
/ and type SQRT and a set of parentheses. Within the parentheses enter the n value (the
number of students that provided data).
9.) Create a Microsoft Excel column graph to compare the average erythrocyte diameter of each
blood treatment group. Include the data from your group on this graph so that individual
results can be compared to the class data. Write a proper figure legend and conclusion on
this graph and identify which of the unknown NaCl solutions was hypertonic and which was
hypotonic relative to the erythrocyte cytoplasm.
10.) Add SEM bars to the columns of data. Right click on your graph, select data, switch
rows/columns. Click on the Plasma column and choose ‘chart layout’ on the top of Excel. Select
errors bars, choose error bars options, select custom and specify value. A pop up window will
appear called ‘custom error bars’. In both the positive and negative error value click on the

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Osmoregulation 59

cell within the spreadsheet for the SEM of visual data. Repeat these steps for the Unknown I
and II data.
11.) You will notice that SEM bars were added to your groups data. These need to be removed. To
do so, right click on the error bar and select format error bar. Choose the end style, no cap.

Group Assignment Due Today:


1.) An XY Scattergraph illustrating the results from Experiment 1 comparing water loss or
gain, as percent of initial mass, in the earthworms the data provided to you. Show the
calculation for the unknown.
2.) A column graph illustrating the results from Experiment 2 comparing your group data
with the class averages.

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to
include your group number, lab day, all names (first and last) and Student ID# from your lab
group members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Question to ponder: The blood plasma osmolality from an average human is


282mOsm, while sea water is 1000mOsm. Using your knowledge of penetrating and non-
penetrating solutes as well as hypotonic, isotonic and hypertonic solutions explain why from
a physiological perspective, it would be bad for humans to drink sea water?

Readings from The Scientific Literature: Through the link below or on reserve in the Natural Science
Library

Strange, K. 2004. Cellular volume homeostasis. Advances in Physiological Education 28: 155-
159. Click here for download

Willmer, P. 2006. Osmoregulation in invertebrates. In: “ENCYCLOPEDIA OF LIFE SCIENCES”


John Wiley & Sons, Ltd: Chichester doi: 10.1038/npg.els.0003646
Click here for download

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Respiratory Physiology 60

LAB EXERCISE #6: HUMAN RESPIRATORY


PHYSIOLOGY
Recommended Textbook Reading:
Sections 49.3 – 49.4 of Russell et al. (2016) Canadian edition
Sections 40.3 - 40.5 of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION
In order to survive, actively metabolizing cells must be supplied with O2 whereas waste CO2
must be removed. In very small animals consisting of a few cell layers, simple diffusion is sufficient
to allow for the exchange of gases between the environment and the internal regions of the
organism. In larger animals, however, the distance between the environment and internal body is
too large for diffusion alone to supply tissues with O2 and remove excess CO2. Larger animals,
therefore, usually have specialized structures to facilitate the exchange of gases. Gills and lungs
greatly increase the surface area over which gaseous exchange can take place. In humans, for
example, the surface area of the lungs is 50 to 100 m2; the rest of the body surface occupies a total
area of less than two m2.
The human respiratory system consists of a series of tubes that branch and terminate as
clusters of small membranous air sacs called alveoli. Oxygen and CO2 rely on simple diffusion to cross
the alveolar membrane. Factors that influence diffusion of gases between lungs and the blood
include the amount of respiratory surface area, diffusion distance and concentration gradient. The
total area of the alveoli is about the size of a tennis court, and their thin walls provide a short
diffusion distance. A high concentration gradient is ensured by (1) movement of blood with low O2
and high CO2 levels to the lungs and (2) pulmonary ventilation (breathing), which maintains a high
level of O2 and a lower level of CO2 in the alveolar air.
Measurements of the amount of air entering and leaving the lung provide valuable
information on the physiology of respiration in humans (Figure 1). The amount of air that moves in
or out of the lungs during any one breathing cycle is called the tidal volume (TV). After normal
inspiration, it is possible to breathe in additional air—this is called the inspiratory reserve volume
(IRV). Similarly, after a normal expiration, it is possible to exhale additional air from the lungs—this
is the expiratory reserve volume (ERV). Even if the expiratory reserve volume is fully expelled from
the lungs, there is still a volume of air in the lungs, called the residual volume (RV) that cannot be
exhaled. The RV has lower O2 and higher CO2 concentrations than atmospheric air. Upon inspiration,
however, fresh air mixes with stale air from the RV, and the alveolar O2 and CO2 concentrations will
be sufficient to facilitate diffusion of the gases to and from the capillaries.
The amount of air moving to and from the respiratory surface of the lungs can be adjusted to
meet the metabolic demands of the individual. This is done by altering the TV as well as increasing
the frequency of breathing. The maximal volume of air that can be exchanged during a single
breathing cycle is called the vital capacity (VC) and represents the maximum TV possible for an
individual. In this situation, all of the IRV and ERV are used to generate the maximum volume of air
exchange in the lung. Adjustments in the TV and breathing rate are regulated by the brain’s

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BIOL 224: Human Respiratory Physiology 61

respiratory center in the medulla oblongata. Peripheral chemoreceptors to detect blood pH, O2, and
CO2 levels, and stretch receptors in the lungs provide important sensory information that modulates
the activity of respiratory center neurons. A component of the human breathing pattern is under
conscious control, although the respiratory center will eventually override these conscious efforts
and ensure that blood gas and pH levels are regulated appropriately.
The quantity of air exchanged during lung ventilation can be measured using a volume
recorder called a spirometer, and these measurements can be used to divide the volume of air
contained within the lung following inspiration and expiration into several components (Figure 1). In
this lab you will measure these parameters in human subjects at rest and immediately after exercise,
when the body’s demands for O2 have been elevated and additional CO2 from muscular metabolic
activity must be eliminated.

Figure 1. Diagram illustrating human lung volumes measured during several breathing
cycles of inspiration and expiration, while at rest and following a maximal forced
inspiration and expiration. The various components of the total lung capacity (TLC) are
labeled as follows: IC – inspiratory capacity; IRV – inspiratory reserve volume; FRC -
functional residual capacity; ERV – expiratory reserve volume; TV – tidal volume; VC -
vital capacity; RV – residual volume. Note that lung volumes do not overlap, but that
lung capacities may contain two or more of the primary volumes. All of the lung
volumes depicted in Figure 1 can be measured using a spirometer, with the exception
of the RV, which must be determined using more sophisticated methods.

EQUIPMENT REQUIRED
PC Computer and iWorx/214 data acquisition unit
SP-304 Spirometer
FH-300 Spirometer flow head and plastic tubing
Disposable cardboard mouthpiece
Nose clip
Tape measure

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BIOL 224: Human Respiratory Physiology 62

EQUIPMENT SETUP & CALIBRATION PROCEDURES


Start the Software
1.) Turn on iWorx214 and open LabScribe. No device founds? Tools, find hardware.
2.) In the Main window, pull down the Settings menu. Select Biology 224 folder and select
the Breathing settings file. No settings file? Lab 1, page 15.
3.) A LabScribe window will appear as configured by the Breathing settings.

Spirometer Setup
1.) Find the SP-304 spirometer, FH-300 flow head and airflow tubing on your bench (Fig. 2).
2.) Firmly push the two airflow tubes onto the two outlets on the FH-300.
3.) Firmly push the other ends of the two airflow tubes onto the outlets of the SP-304 unit.
4.) Plug the DIN8 connector from the SP-304 into the Ch4 input of the iWorx/214 (Fig. 2).

Figure 2. The SP-304 Spirometer, FH-


300 flowhead, and the airflow tubing
used to collect data on human lung
volumes.

Spirometer Calibration
The Breathing settings file configures LabScribe to record the breathing of the subject on the Air
Flow channel. However, the computed function in the Volume channel converts the data recorded
on the Air Flow channel to actual lung volume measurements. The following steps are used to
calibrate the SP-304 spirometer for this data conversion.
1.) In the main LabScribe Window, click on the words Vol. Human (AirFlow), which are next to
the title of the C1 Lung Volume channel, to open a pull-down menu.
2.) Select Setup Function from this pull-down menu to open the Spirometer Calibration
Dialog window as seen in Figure 3.
3.) Enter the calibration voltage (listed on the label on the bottom of your SP-304 spirometer)
into the equation that sets the calibration voltage equal to one liter of lung volume.
4.) Make sure Reset After 60 seconds is selected, and the first 5 seconds of the recording are
used to zero the baseline of the Volume channel. Click OK.

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BIOL 224: Human Respiratory Physiology 63

Enter the hand-written


calibration value located on
the under-side of the SP-
304 in the input box
between 1 Liter = and volts.

Figure 3. Spirometry calibration window.

Functional Equipment Test


Allow the SP-304 to warm up for 10 minutes before recording for the first time. Note: Do not hold
the SP-304 spirometer in your hand; the heat from your hand will alter the volumes recorded.
*Your volunteer must not have a pre-existing medical condition such as a history of cardiovascular
or respiratory problems.
1.) Attach a disposable cardboard mouthpiece to the flowhead and determine if your volunteer
is breathing through the correct end of the flowhead. If not, place the cardboard
mouthpiece on the other side of the flowhead. When spirometry data is recorded in the
conventional manner, inhalation is always displayed as an upward deflection.
2.) Click on the Record button.
3.) Wait 5 seconds before the subject inhales through the disposable mouthpiece attached to
the flowhead. Note: The LabScribe software will zero the Volume channel during the
first five seconds of recording. No air should be moving through the flow head during this
time.
4.) Click on the AutoScale button at the upper margin of the Air Flow and Volume channels.
5.) If the flowhead is oriented properly, the traces on the Air Flow and Volume channels will go
up during inhalation.
6.) If the traces on these channels go down during inhalation, have the subject breathe through
the other end of the flowhead.
7.) Click on the Stop button.

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BIOL 224: Human Respiratory Physiology 64

EXPERIMENT: RESPIRATORY VOLUMES DURING REST AND AFTER MILD EXERCISE

Before Starting
1.) Please read the procedures for each exercise completely before beginning the experiment.
You should have a good understanding of how to perform these exercises before
making recordings. There are a large number of calculations to be performed. Wherever
possible, divide these calculations between the various members of your group so that
the calculations are done in an efficient manner.
2.) The spirometer will monitor breathing from a subject. It is important that the subject is
healthy and has no history of respiratory or cardiovascular problems.
3.) On the flow head, the outlets connected to the airflow tubing should always be pointed up to
avoid problems with condensation developing within the tubing.
4.) To reduce turbulence within the flowhead, place a disposable cardboard mouthpiece over
the opening of the flowhead. Use a new mouthpiece for each subject.
5.) Use a nose clip to prevent air from entering or leaving the nose as the subject is breathing.
Air that passes through the nose is not included in the volume measurements and
causes errors in these values.
6.) Remember that the LabScribe software will zero the Volume channel during the first five
seconds of recording. No air should be moving through the flow head during this time.
Lung Volume Measurements While Subject Is Resting
1.) The subject should sit comfortably with their feet on the floor and become accustomed to
breathing through the flowhead with a nose plug in place. Have the subject hold the
flowhead so that its outlets are pointed up and that the head is slightly tilted back.
2.) Click Record. Do not breathe through the flowhead for the first 5 seconds of the
recording to allow the volume channel to zero. Have the subject practice breathing
normally through the flowhead for a few minutes.
3.) Type Resting in the Mark box that is to the right of the Mark button. Press the Enter key on
the keyboard to mark the recording.
4.) Click the the AutoScale buttons of the Air Flow and Volume channels and Double Display
Time about 4 or 5 times. Notice the slowly moving wave on the Volume channel.
5.) Record five breaths, which normally takes about forty-five seconds. This recording will be
used to measure the subject's tidal volume (TV).
6.) Type Forced in the Mark box. Press the Enter key on the keyboard as the subject inhales as
deeply as possible. After reaching his or her maximum inhalation volume, the subject
should exhale forcibly and maintain the exhalation for about a second. This recording
will be used to measure the subject's vital capacity (VC).
7.) Adjust your recording so it looks similar to Figure 4. Repeat steps 5 and 6 two more times.

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BIOL 224: Human Respiratory Physiology 65

8.) Stop recording and save the data as <Subjects Name>. Choose a destination on the
computer like the V:drive. Designate the file type as *.iwxdata. DO NOT CLOSE THIS
FILE.

Figure 4. Typical LabScribe


recording of airflow (top
window) and lung volume
(bottom window) during normal
and forced breathing of a human
subject at rest.

Lung Volume Measurements After Mild Exercise


1.) Have the subject remove the flowhead mouthpiece and take off the nose plug. Ask him or
her to engage in five to ten minutes of mild exercise. Depending on their level of fitness,
this can be something like deep knee bends, stair climbing or a jog around the bowl
(weather permitting, of course). Walk, do not run the stairs.
2.) The subject should return to the lab room immediately after their mild exercise, and sit
comfortably with their feet on the floor. This and all following steps should be done as
quickly as possible after exercise.
3.) Click on the Record button. After waiting five seconds for the Volume channel to zero, have
the subject place the flowhead in his or her mouth, put the nose clip back on and begin
breathing through the spirometer.
4.) Type After Exercise in the Mark box to the right of the Mark button. Press the Enter key to
mark the recording.
5.) Click the AutoScale and Double Display Time buttons of the Air Flow and Volume
channels. Record at least five breaths as the subject is recovering from exercise
6.) Type Forced in the Mark box. Press the Enter key on the keyboard as the subject inhales
as deeply as possible. After reaching his or her maximum inhalation volume, the subject
should exhale as completely as possible.
7.) Breath normally for a few tidal volumes and repeat step #6 two more times.
8.) Click Stop to halt recording. Save the data.
Data Analysis: Lung Volumes During Normal Breathing at Rest
1.) Scroll through the recording and find the data recorded when the subject was resting.

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BIOL 224: Human Respiratory Physiology 66

2.) Place the cursors on either side of a group of five complete resting breathing cycles and a
forced breath (see Figure 4). Click the Zoom between Cursors button to expand the
selected breathing cycles to the width of the Main window.
3.) Click on the Analysis window icon in the toolbar
4.) Look at the Function Table that is above the uppermost channel displayed in the Analysis
window. The mathematical functions, V2-V1, and T2-T1 should appear in this table.
5.) Minimize the height of the Air Flow channel by placing the cursor on the dividing line between
the two channels of data and dragging the Volume channel upwards. Measurements will
be taken from the Volume channel.
6.) Maximize the height of the trace on the Volume channel by clicking on the arrow to the left
of the channel’s title to open the Channel menu. Select Scale from the menu and
AutoScale from the Scale submenu to increase the height of the data on that channel.
7.) Use the mouse to click on and drag the cursors to specific points on the Volume channel
recording so that various parameters can be measured as described in Steps 8 & 9
below. Once the cursors are placed in the correct positions, the values shown in the
Function Table can be recorded automatically to the Journal with the Add Ch. Data to
Journal in the Channel pulldown menu.
8.) Place one cursor in the trough prior to the first inhalation and the second cursor on the peak
of this inhalation cycle. The value for the V2-V1 function on the Volume channel is the
tidal volume (Fig. 5). Record this value in the Journal. Repeat this for the next two
cycles. Calculate the average tidal volume from these three measurements. Enter this
value in Table 1.

Figure 5. Breathing pattern of a subject at rest. Figure 6. Normal breathing pattern of a subject at rest,
The cursors are positioned on the trough and displayed on the Volume channel in the Analysis
the peak of the breath cycle to measure the window. The cursors are positioned on the peaks of
tidal volume (TV) with the V2-V1 function. successive breath cycles to measure the breath period
with the T2-T1 function.

9.) Measure the duration of each breathing cycle (breath period). Place one cursor on a peak
of a breath cycle, and the second cursor on the peak of an adjacent cycle. The value for T2-
T1 is the period of that breath cycle (Fig. 6). Record this value in the Journal. Repeat this for
the next two cycles. Calculate the average breath period from these three measurements.

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BIOL 224: Human Respiratory Physiology 67

10.) Calculate the normal breathing rate of the subject at rest using the following equation and
enter this value in Table 1:
Breathing Rate (breaths/minute) = 60 seconds/minute
mean breath period (sec/breath)

11.) Multiply mean tidal volume by breathing rate to calculate the volume of air passing through
the subject’s lungs each minute. Write this minute respiratory volume in Table 1.
12.) Unzoom the data by clicking on the Double Display Time icons in the LabScribe toolbar.
Data Analysis: Lung Volumes from Forced Inhalation and Exhalation at Rest
1.) In the Analysis window, measure the Inspiratory Reserve Volume (IRV) by placing one
cursor on the peak of a normal breath prior to maximum inhalation and the second
cursor on the peak of the forced inspiration. Add the value for the V2-V1 function on the
Volume channel to the Journal.
2.) Measure the Vital Capacity (VC) by placing one cursor on the peak of the forced breath
cycle and the second cursor on the flat line after the subject has expelled all the air from
his or her lungs. Add the value for the V2-V1 function on the Volume channel to the
Journal.
3.) Measure the Expiratory Reserve Volume (ERV) by placing one cursor in the trough before
maximal inhalation and the second cursor on the flat line after subject has expelled all
the air from his or her lungs. Add the value for the V2-V1 function on the Volume
channel to the Journal. Take the absolute value.
4.) Calculate the average from the 3 trials and write the IRV, VC and ERV measurements in
Table 1.
Data Analysis: Lung Volumes Immediately After Mild Exercise
1.) Scroll to the data collected immediately after mild exercise.
2.) Repeat the same measurements as were performed for breathing at rest and forced
inhalation/exhalation at rest. Find the average and Add the values to your Journal.
3.) Record the various post-exercise values in Table 1.
Data Presentation
1.) Draw an Excel column graph showing your subject’s TV, IRV, ERV & VC while resting and
after mild exercise.
Data Analysis: Estimated Residual Volume Calculation
1.) Since it is not possible to measure Residual Volume (RV) using the spirometer, the RV of
your subject must be estimated. In healthy individuals, the VC is approximately 75%
of the Total Lung Capacity (TLC).
2.) Use this assumption and the resting VC value from Table 1 to calculate the TLC of your

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BIOL 224: Human Respiratory Physiology 68

subject. The estimated RV is the difference between the predicted TLC and the resting
VC.
3.) Record the estimated RV value in Table 1.
Data Analysis: Calculation of Lung Capacities
1.) The lung volume data from Table 1 can now be used to calculate various lung capacities.
2.) Use the various formulae in Table 2 to calculate these for your subject.
3.) Provide these values to your lab demonstrator who will then calculate class averages. Write
the class average values in Table 2 when these are available.
Data Presentation
1.) Use the class averages in Table 2 to draw an Excel column graph comparing the various
lung capacities in men and women.
Data Analysis: Predicted Vital Capacity Calculations
Clinicians often make use of various formulae that have been developed over the years to predict
the expected lung volumes in patients. Actual spirometry measurements of lung volumes in patients
are then compared to the predicted values. This comparison can be used to assess overall lung
function in patients. Significant deviations from the predicted values may represent some sort of a
disease state. However, there are many factors that influence lung volume in individuals and the
predictive formulae do not always provide an accurate estimate for an individual patient. It is
generally assumed that measured volumes within 20% of the predicted volume are considered
normal. Differences greater than 20% are not, in and of themselves, diagnostic of a lung disease but
simply point to the need for more sophisticated testing. The predictive power of a simple formula
sometimes used to estimate VC in patients will be examined in this part of the lab exercise. Keep in
mind that more sophisticated formulae are generally used in real clinical situations.
1.) A rough estimate of the VC in liters can be calculated from the following formulas based on
height (H) in centimeters and age (A) in years:
VC (Male subject) = (0.052H) - (0.022A) - 3.60
VC (Female subject) = (0.041H) - (0.018A) - 2.69
2.) Use the tape measure to determine the height of your subject (without shoes).
3.) Use the appropriate formula above to calculate the predicted vital capacity of your subject.
4.) Record this predicted value in Table 3 below. Also include the measured vital capacity (from
Table 1) of your subject in the table below.
5.) Your teaching assistant will collect similar values from five of your neighbouring lab groups
for you. Write these values in Table 3.
Data Presentation
1.) Draw an Excel line graph comparing measured and predicted VC values from Table 3. Make
sure you put your data in ascending order before you create your graph.

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BIOL 224: Human Respiratory Physiology 69

Group Assignment Due Today:


1.) An Excel column graph utilizing data in Table 1.
2.) An Excel column graph utilizing data in Table 2.
3.) An Excel line graph utilizing data in Table 3.
4.) The Group Data Tables from page 10. Be sure to show the ‘at rest’ calculations for
residual volume, breathing rate and minute respiratory volume determined for Table 1.
Show a calculation for predicted vital capacity.

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to
include your group number, lab day, all names (first and last) and Student ID# from your
lab group members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Question to ponder: Give a physiological reason that explains the relationship


between lung size and body size.

Readings from the Scientific Literature: Through the link below or on reserve in the Natural Sciences
Library
Clayton, N. 2007. Assessing lung size. Chronic Respiratory Disease 4: 151–157
Click here for download

Guyton, A.C. and J.E. Hall. 2006. Pulmonary Ventilation. In Textbook of Medical Physiology (eds
W. Schmitt & R. Gruliow). pp. 471-482. Philadelphia, PA. Elsevier Inc.

Guyton, A.C. and J.E. Hall. 2006. Regulation of Respiration. In Textbook of Medical Physiology
(eds W. Schmitt & R. Gruliow). pp. 514-523. Philadelphia, PA. Elsevier Inc.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Respiratory Physiology 70

BIOL 224 Lab Exercise #6 – Group Data Tables


Group Members:
Table 1: Measured and Calculated Lung Function Parameters From Experiment 1
Lung Function Parameter While At Rest After Exercise
Tidal Volume (TV)
(in liters)
Inspiratory Reserve Volume (IRV) (in
liters)
Expiratory Reserve Volume (ERV) (in
liters)
Vital Capacity (VC)
(in liters)
Residual Volume (RV) (estimate)
(in liters)
Breath period (seconds)
Breathing Rate
(breaths/minute)
Minute Respiratory Volume
(liters/minute)

Table 2. Calculated Lung Capacities


From Your Class Average Class Average
Parameter
Subject At Rest For Women For Men
Inspiratory Capacity
(IC) = TV + IRV
Expiratory Capacity
(EC) = TV + ERV
Functional Residual Capacity
(FRC) = ERV + RV
Total Lung Capacity
(TLC) = TV + RV + IRV + ERV
Table 3. Comparison of Measured and Predicted Vital Capacities
Measured Vital Capacity (liters) Predicted Vital
Capacity (liters)
Your Subject
Subject #2
Subject #3
Subject #4
Subject #5
Subject #6

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Circulatory System Physiology 71

LAB EXERCISE #7: HUMAN CIRCULATORY


SYSTEM PHYSIOLOGY
Recommended Textbook Reading:
Sections 40.3 – 40.5 of Russell et al. (2016) Canadian edition
Sections 41.3 – 41.5 of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION
The cardiac cycle in humans and other vertebrates involves the sequential contraction of the
atria and the ventricles. The heart’s rhythmical contraction sequence is triggered by action potentials
from myocardial cells that are conducted in a coordinated fashion throughout the entire heart.
Bodily fluids are excellent conductors of electricity and electrical activity in the interior portion of
the body can be easily recorded at the body surface. For example, electrical currents associated with
the depolarization and repolarization of the heart spread easily into the tissues surrounding the
heart. Using electrodes placed on opposing sides of the heart, this electrical current can be measured
as an electrical potential across the body surface, and displayed over time as an electrocardiogram
(ECG). A typical human ECG is shown in Figure 1. The measurement and interpretation of ECG in
humans and other animals play very important roles in the clinical diagnosis of cardiac arrhythmias
and other types of heart damage.
Figure 1: A typical human ECG
recorded with LabScribe. Components
of the ECG correlated with electrical
activity in the atria and ventricles are
labeled as follows:
P wave - atrial depolarization;
QRS complex - atrial repolarization
and ventricular depolarization;
T wave - ventricular repolarization
Note: There is no distinct wave
representing atrial repolarization in
the ECG because it occurs during
ventricular depolarization (much
larger amplitude).

Although electrical signals to initiate vertebrate heart contraction originate in the myocardial
cells, several aspects of heart activity can be modified by the autonomic nervous system. This
nervous system input is used to adjust cardiac function to meet the immediate needs of body tissues.
Excitation or inhibition of the heart is accomplished by changes to the contraction rate and various
other parameters associated with myocardial contraction. An obvious example is the increase in
heart rate that occurs during exercise. On an ECG, a change in heart rate can be easily measured as
a change in the P-P interval. Increased heart rate (measured in beats per minute) is primarily
accomplished by reducing the time between beats (i.e. the T-P interval) and the overall time that a
complete depolarization/repolarization cycle occurs (i.e. the P-T interval). This latter effect is

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BIOL 224: Human Circulatory System Physiology 72

accomplished by altering the conduction velocity of the electrical signals as they travel throughout
the heart.
Once ejected from the heart, blood enters the arterial system for distribution throughout the
body. The arterial system functions as a pressure reservoir in that the amount of blood flow is
directly related to the pressure difference along an artery. Increased heart function will increase
centralized blood pressure, but signals from the autonomic nervous system can also control the
degree of contraction of the smooth muscle found in the walls of the arteries. Vasoconstriction of
the arteries will increase centralized blood pressure whereas vasodilation has the opposite effect.
In addition to playing an important role in blood pressure regulation, autonomic nervous system
effects on the arterial system can be used to alter the distribution of blood to various organs in the
body depending on the metabolic needs of the animals. For example, blood flow to the gut decreases
during exercise, while blood flow to the skeletal muscles increases dramatically.
The autonomous nervous system can also directly influence heart rate. In the fight or flight
response, the sympathetic nervous system releases epinephrine from the adrenal glands, which
binds to receptors on the heart causing it to beat faster. On the other hand, parasympathetic release
of acetylcholine from the vagus nerve binds to receptors on the heart causing it to beat slower. Any
change in heart rate has the potential to alter arterial blood pressure, especially in central arteries
such as the aorta. For example, while vasodilation of arteries supplying skeletal muscle during
exercise has the potential to cause a drop in blood pressure elsewhere in the body, an increase in
heart rate while cause an increase in pressure. Several peripheral feedback loops within the
autonomic nervous system are used to simultaneously coordinate heart activity, arterial blood
pressure and overall blood flow in the body. The baroreceptor reflex is one of the most important
of these feedback loops. This reflex normally acts to ensure that central arterial blood pressure is
maintained at a level appropriate for metabolic activities in the body, but that is not too high to
cause rupture of arterial vessels or excess fluid leakage from the capillaries.
Diving in humans and other air-breathing animals is another situation where the
baroreceptor reflex is used to coordinate heart activity, arterial blood pressure and peripheral blood
flow. Diving in these animals generally causes selective peripheral vasoconstriction and a sharply
reduced blood flow to the limbs, gut and the skin. This ensures that blood is delivered to organs with
the highest need for oxygen, including the brain and heart. However, the selective vasoconstriction
that takes place during diving has the potential to cause a significant increase in the blood pressure
of the central arteries. This is avoided by a baroreceptor-mediated response known as diving
bradycardia, where heart rate is substantially reduced to ensure that blood pressure in central
arteries does not exceed safe levels.
In this lab period, you will record and measure a number of parameters associated with the
physiology of the human circulatory system, including the effects of exercise and a simulated dive
on ECG and peripheral blood flow. If time permits, you can also use a stethoscope to listen to sounds
generated as the heart contracts and measure arterial blood pressure with a sphygmomanometer.

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BIOL 224: Human Circulatory System Physiology 73

Equipment & Materials Required


PC Computer and the iWorx/214 data acquisition unit
C-AAMI-504 ECG cable and electrode lead wires
PT-104 Pulse plethysmograph
Alcohol swabs
Disposable ECG electrodes
Dish basin to be filed with tap water for the diving experiment
Stethoscope
BP-600 Non-invasive blood pressure transducer

BEFORE STARTING
1). Please read the procedures for each exercise completely before beginning the experiment.
You should have a good understanding of how to perform these exercises before making
recordings. There are a number of calculations and measurements to be performed.
Wherever possible, divide these calculations between the various members of your group
so that the calculations are done in an efficient manner.
2). It is important that the subjects volunteering in this exercise are healthy and have no
history of respiratory or cardiovascular problems.

START THE SOFTWARE


1.) Turn on iWorx 214 and open LabScribe. No device found? Tools, find hardware.
2.) On the Main window, pull down the Settings menu. Select the ECG Settings file from
the Biology 224 folder. No settings folder? Lab 1, page 12.

EQUIPMENT SET-UP
1). Locate the C-AAMI-504 cable, electrode lead wires and pulse plethysmograph (Fig. 2).

Figure 2: The C-AAMI-504 cable, electrode lead


wires and PT-104 pulse plethysmograph
connected to an iWorx 214 data acquisition
unit.

2). Insert the black connector on the end of the C-AAMI-504 cable into the Isolated Inputs
of Ch 1 & 2 of the iWorx/214 unit. Plug the PT-104 pulse plethysmograph into the
Non-Isolated Ch 3 input.

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BIOL 224: Human Circulatory System Physiology 74

3). Insert the connectors on the red, black, and green electrode lead wires into the
matching sockets on the lead pedestal of the cable. Do not plug in the white or
brown.
4). Instruct your volunteer to remove all jewelry from their wrists and right ankle.
5). Use an alcohol swab to clean and scrub a region with little or no hair on the inside of
the subject’s right wrist. Let the area dry. SCREW THE LID BACK ONTO THE TOP OF
THE RUBBING ALCOHOL BOTTLE.
6). Remove a disposable ECG electrode from its plastic shield, and apply the electrode to
the scrubbed area on the wrist.
7). Repeat Steps 6 and 7 for the inside of the left wrist and the inside of the right ankle.
8). Snap the lead wires onto the electrodes, so that:
• the red (+1) lead is attached to the right wrist or just below the right clavicle,
• the black (-1) lead is connected to the left wrist or just below the left clavicle,
• the green (G or ground) lead is connected to the right inner ankle.
9). Place the plethysmograph on the volar surface (where the fingerprints are located) of
the distal segment of the left middle finger, and wrap the Velcro strap around the end
of the finger to attach the unit firmly in place.
10). Instruct the subject to sit quietly with their hands palm-up on the lab bench and feet
on the floor. If the subject moves, muscle electrical activity will disrupt the ECG
recording.

FUNCTIONAL EQUIPMENT TEST


1). Click on the Record button. Obtain a minute or so of recording. Click Stop.
2). Click on the AutoScale button at the upper margin of the channels. Your recording
should look similar to that in Figure 3.
3). If either signal is upside down when compared to the traces in Figure 3, click on the
downward arrow to the left of the channel title and select the Invert function.
4). All waves of the ECG should be clearly visible. If they are not, remove the electrodes
and rescrub the skin with additional alcohol. Also, ask your volunteer to sit as quietly
as possible.
5.) If pulse is not visible after auto-scaling, reattach the plethysmograph or try the middle
finger of the opposite hand.

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BIOL 224: Human Circulatory System Physiology 75

Figure 3. LabScribe main window showing representative recordings of ECG (top)


and finger pulse (bottom) data from a human volunteer.

Experiment #1: Effects of Exercise on the ECG and Peripheral Circulation


1). When you have a suitable trace, type Resting ECG in the Mark box. Click Record
and press the Enter key on the keyboard to attach the comment to the data. Record
for a minute or two.
2). Click Stop to halt recording.
3). Select Save As in the File menu, type a name for the file and save to your V: drive.
4). Disconnect that lead wires from the ECG electrodes (do not pull the electrodes off the
skin) and unplug the pulse plethysmograph from the iWorx 214 and wrap it around
your hand.
5). Ask your volunteer to perform 5 to 10 minutes of mild exercise. If you choose to climb
stairs, walk up and down the stairs, do not run the stairs.
6). Immediately after the exercise period is over, snap the lead wires back into place on
the skin electrodes and reattach the pulse plethysmograph. Click Record.
7). Mark this recording as ECG-Recovery from Exercise.
8). Continue recording for five minutes.
9). Click Stop to halt recording. Save the data.
Data Analysis For Experiment 1
1). Scroll through the recording and find a section of data with three exemplary ECG
cycles recorded in succession while the subject was resting.
2). Select three adjacent ECG cycles with the double cursors Click the Zoom between
Cursors button to expand the segment to the width of the Main window and click on
the Analysis window.

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BIOL 224: Human Circulatory System Physiology 76

3). An example ECG recording with lines showing the correct placement of cursors at the
start of the P waves is shown in Figure 4 below.

Figure 4. An example human ECG with lines showing the start of the P waves where
cursors are to be placed to measure various ECG intervals. If reading is noisy you may
place the cursors on the peak of each wave to measure the ECG intervals.
4). On your ECG recording, use the double cursors to measure the following intervals on
each of the three successive ECG cycles:
• P-T interval
• T-P interval
• P-P interval
5). Click on the downward arrow beside the ECG channel name. Select Add channel
data to Journal. Keep track of the interval names and the corresponding time
information.
6). Calculate the average (mean) for each interval. Write this information in Table 1 on the
Group Data Table sheet on page 9.
7). The P-P interval measures the time needed for one heart beat. Use the mean P-P
interval to calculate heart rate in beats per minute using the following equation:
Heart Rate (beats/minute) = 60 seconds/minute
seconds/beat

8). On the Pulse window, position the first cursor on the onset of the pulse wave and the
second cursor on the peak of each pulse wave that corresponds to the ECGs measured
above. Click on the downward arrow beside the Pulse channel name. Select Add
channel data to Journal. Calculate and write the average of the amplitudes in Table 1.
[Pulse height reflects peripheral blood flow and would normally have units other than
mV. However, calibration of the pulse plethysmograph is time consuming and has
been omitted for convenience.]
9). Scroll through the recording and find a section of data with three exemplary ECG and
pulse cycles recorded in succession immediately exercising. Repeat steps 2 through
10 described above EXCEPT OMIT FINDING THE P-T and T-P INTERVALS. Write all data
in Table 1.
10). Repeat these steps for data recorded one minute and four minutes after exercise.

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BIOL 224: Human Circulatory System Physiology 77

Data Presentation for Experiment 1


1). Draw a double y-axis line graph of your subject’s heart rate and pulse height recorded at
rest, immediately after exercise, and one and four minutes after exercise. Heart rate
and pulse height need to be graphed on separate axes as their units are completely
different.

Help for creating a double y-axis graph:


In an excel spreadsheet organize your columns with headings as follows:

Activity Heart Rate Pulse Amplitude

Enter the data under the corresponding headings and create a line graph. Finish the figure as a
new chart. Right click on one of the lines of data in the figure. Select ‘Format Data Series’. Click
on secondary axis. You should now see that there are gradations on both the left and right side
of the graph. Make sure both y-axes are labeled.

Experiment 2: Heart Rate and Peripheral Blood Flow During Diving


The experimental set-up is identical to experiment 1.
1.) On the Main window, pull down the Settings menu. Select the ECG settings file from the
Biology 224 folder. A second volunteer should be used for this experiment. Hint: an
experienced swimmer or diver will likely show a very good diving response.
2.) Using cool tap water (not too cold) fill the dish basin 2/3 full. Make sure that there are paper
towels available for the volunteer. Place the basin as far away from the recording unit
and computer as possible.
3.) Perform the equipment set-up of the ECG cables and pulse plethysmograph as described in
experiment 1. Make sure clear ECG and pulse data are being recorded.
4.) Instruct the subject to stand quietly in front of the basin. Have one member of your group
stand by with paper towels and otherwise assist your volunteer. Remind the volunteer to
keep the pulse plethysmograph out of the water. Also remind her/him to keep as still as
possible during the various sections of the experiment.
5.) Click Record. You will record continuously for the duration of the experiment.
6.) Type pre-dive as a comment in the Mark box. Record for 20 to 30 seconds. Ensure that you
have fairly clear data tracings.
7.) Type dive into the Mark box and have the subject take a deep breath and submerge their
face (up to the hairline and in front of the ears) in water for 20-30 seconds.
8.) When the subject removes their face from the water type surface in the Mark box and
continue to record for another two minutes.
9.) Stop recording. Save the data to the V:drive.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Circulatory System Physiology 78

Data Analysis For Experiment 2


1.) Scroll through your data and find sections of the data set containing three exemplary and
successive ECG cycles in each of the following sections of the recording:
• a stable area shortly before facial immersion.
• near the first five seconds following facial immersion.
• near the last five seconds of facial immersion.
• near the first five seconds after face is removed from water.
• near the last five seconds at the end of recording.
2.) In the Analysis Window, determine average the P-P interval, which is the time for one
heartbeat, average heart rate, and average pulse amplitude within each section of data
listed above. To measure pulse amplitude, position the first cursor on the onset of the
pulse wave and the second cursor on the peak of each pulse wave that corresponds to
the ECGs measured above.
3.) Write these values in Table 2 on the Group Data Tables sheet on page 11.
Data Presentation for Experiment 2
1.) Draw a double y-axis line graph of your subject’s heart rate and pulse height recorded at the
various times during Experiment 2.
Experiment 3 (Optional): Heart Sounds and Arterial Blood Pressure Measurement
If time permits, use the stethoscope and sphygmomanometer provided to listen to the hearts
sounds and obtain arterial blood pressure readings from your lab partners. The hearts sounds “lub
dub” are made as valves within the heart close. As the valves close, a very small amount of blood is
pushed backward through the valve. This creates a very short period of turbulence that can be heard
through the stethoscope. Thus, listening to the heart sounds can provide considerable information
about overall heart and valve function. The sphygmomanometer is the most commonly used device
to determine arterial blood pressure. Instructions for using the sphygmomanometer will be provided
in the lab period.

Group Assignment Due Today:


1.) An Excel double y-axis line graph utilizing data (heart rate and pulse amplitude) in Table 1.
2.) An Excel double y-axis line graph utilizing data in Table 2.
3.) The Group Data Tables. Show a heart rate calculation.

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to include
your group number, lab day, all names (first and last) and Student ID# from your lab group
members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Circulatory System Physiology 79

Question to ponder: Refer to the definitions of the baroreceptor reflex and diving
bradycardia. How would the feedback loop of the baroreceptor reflex help explain why
some people survive after falling into cold water and being submerged for long periods of
time?

Readings from the Scientific Literature: Through the link below or on reserve in the Natural Sciences
Library
Andersson, J., E. Schagatay, A., Gislén and B. Holm. 2000. Cardiovascular responses to cold-water
immersions of the forearm and face, and their relationship to apnoea. Eur. J. Appl. Physiol.
83: 556-572 Click here for download
Ferrigno, M., G. Ferretti, A. Ellis, D. Warkander, M. Costa, P. Cerretelli, and C.E.G. Lundgren. 1997.
Cardiovascular changes during deep breath-hold dives in a pressure chamber. J. Appl. Physiol.
83: 1282-1290. Click here for download
Fadel, P.J an P.B. Raven. 2011. Human investigations into arterial and cardiopulmonary
baroreflexes during exercise. Exp. Physiol. 97: 39-50 Click here for download
Levy, M.N., and A. Pappano. 2006. Interplay of central and peripheral factors in control of the
circulation. In Berne and Levy Principles of Physiology (eds W.R. Schmitt and A. Hall) pp. 346-
356
Lindholm P, C.E.G Lundgren. 2009. The physiology and pathophysiology of human breath-hold
diving. J. Appl. Physiol. 106: 284-292 Click here for download
Raven, P.B. 2008. Recent advances in baroreflex control of blood pressure during exercise in
humans: an overview. Medicine & Science in Sports & Exercise. 40: 2033-2036
Click here for download
Raven, P.B., P.J. Fadel, S. Ogoh. 2006. Arterial baroreflex resetting during exercise: a current
perspective. Ex. Physiol. 91: 37-49 Click here for download

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Human Circulatory System Physiology 80

BIOL 224 Lab Exercise #7 – Group Data Tables


Group Members:
Table 1: ECG & Pulse Amplitudes From Experiment 1
While At Immediately 1 minute 4 minutes
Parameter Rest After Exercise After Exercise After Exercise

N/A N/A N/A


Mean P-T interval
(seconds)
N/A N/A N/A
Mean T-P interval
(seconds)

Mean P-P interval


(seconds)

Heart Rate
(beats per minute)

Mean Pulse
Amplitude (mV)

Table 2: ECG Intervals, Heart Rate and Pulse Amplitudes from Experiment 2
Parameter Before Start of End of After End of
Diving Diving Diving Surfacing Recording

Mean P-P
interval
(seconds)

Heart Rate
(beats per
minute)

Mean Pulse
Amplitude (mV)

Heart Rate Calculation:

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Metabolism 81

LAB EXERCISE #8: METABOLISM


Recommended Textbook reading:
Sections 50.5 and 50.6 of Russell et al. (2016) Canadian edition
Sections 42.5 – 42.6 of Russell et al. (2018) Canadian edition

BACKGROUND INFORMATION

Metabolism is the sum of all of the chemical reactions that occur in an organism. These reactions
can be both catabolic and anabolic in nature. In catabolic (destructive) reactions, large organic
molecules are broken down into smaller molecules, a process that usually releases energy (usually
in the form of ATP). In anabolic (constructive) reactions, small precursor molecules are assembled
into larger organic molecules, a process that requires the input of energy (often as ATP). Metabolism
is a constant process that begins with the inception of life and ends with the death. Thousands of
metabolic reactions occur at the same time and all are strictly regulated to keep cells healthy and
working.
Metabolic rate can be defined as the amount of energy used by an animal within a specific period
of time. The maintenance of metabolic rate requires a steady supply of energy to meet the
fluctuating demands. The energy is produced by the controlled oxidation of cellular fuels
(carbohydrate, lipids and proteins), a process known as cellular respiration. In short, oxygen and an
organic molecule react to produce water, carbon dioxide and energy. Therefore, the amount of
oxygen consumed per unit time can be used to calculate the metabolic rate of an animal. In closed
system respirometry, an animal is confined to a closed, water or air-filled, chamber in which the
amount of oxygen consumed is measured over designated periods of time. Oxygen consumption is
revealed by successive determinations of the decreasing amount of oxygen dissolved in the water or
present in the air. The measurements can be performed by using an oxygen electrode or through
chemical titration.
Two major factors influence the metabolic rate of an animal: body temperature and body size.
Other important factors include physical activity, diet, general state of health, and hormones such
as those secreted by the thyroid gland and the adrenal cortex. The metabolic rate of an animal is
influenced by whether the animal is warm-blooded (endothermic) or cold-blooded (ectothermic).
Endothermic animals have a fairly high rate of metabolism compared ectothermic animals. In
addition, another important determinant of metabolic rate is the body weight of the animal. The
metabolic rate in individuals increases directly as a function of their weights (Figure 1), meaning
larger animals consume more oxygen. However, if the metabolic rate is expressed as the rate of
oxygen consumption per unit weight per unit time (e.g. moles O2 consumed/gm of body
weight/hour), the opposite trend is found; this is known as the mass-specific metabolic rate. In other
words, smaller animals consume more oxygen per gram of body weight than larger animals do.
This lab exercise will examine the influence of body weight on the rate of oxygen consumption
in an aquatic ectothermic animal.

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BIOL 224: Metabolism 82

3
10
Endotherms, 39°

Metabolic rate (kcal/h)


0
10

-3
10
Unicellular
organisms, 20°
-6
10
Ectotherms, 20°

-9
10

-12
10

-12 -9 -6 -3 0 3 6
10 10 10 10 10 10 10
Mass (g)

Figure 1. The relationship between Body Weight and Metabolic Rate (expressed as energy
expended per unit time).

Equipment and Materials Required


PC Computer and iWorx 214 data acquisition unit
ISE-730 Dissolved oxygen electrode
DO2-100 Current to voltage adapter, and the DIN-DIN cable
Aquaria with fish (species to be determined) of variable size
Aerated de-ionized, and de-chlorinated tap water
Club soda
125 ml and 250 ml Erlenmeyer flasks with a rubber stopper
100 ml glass beakers
500 mL Graduated Cylinder
Top-loading balance
Aluminum foil

EXPERIMENT: RATE OF OXYGEN CONSUMPTION IN FISH

This experiment will examine the rate of oxygen consumption in fish using a closed-to-air
respiration chamber. The iWorx 214 recording equipment will be fitted with the ISE-730 dissolved
oxygen electrode, and this set-up will be configured to record the decrease in dissolved oxygen
concentration in the water housing the fish over a period of 30 minutes.

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BIOL 224: Metabolism 83

The ISE-730 dissolved oxygen electrode will be prepared for use by the lab demonstrator. Handle
it carefully. The tip of electrode is covered by a delicate Teflon membrane which can tear easily. Do
not tighten or loosen the plastic housing of the electrode, as this may affect its performance.
Each group will be assigned a fish to test in this experiment. Handle the fish carefully and gently.
Remember that you will measure the basal metabolic rate (the amount of energy required to
maintain metabolism while at rest), therefore you must keep the stress level of the fish to a
minimum.

EQUIPMENT SET-UP
1.) Open the LabScribe program. No device found? Tools, find hardware.
2.) On the Main window, pull down the Settings menu and select Biology 224 Metabolism.
3.) The ISE-730 dissolved oxygen electrode (Figure 2) will be fitted with the DO2-100 current to
voltage adapter. The DIN-DIN Cable will connect the voltage adapter to Channel 3 on
the iWorx data recording unit. Make sure that the equipment set-up corresponds to that
shown in Figure 2 & 3.

Figure 2. The ISE-730 dissolved oxygen electrode Figure 3. The ISE-730 electrode and amplifier
(DO2-100) connected to an iWorx 214 with a
male DIN-DIN cable.

CALIBRATION OF DISSOLVED OXYGEN ELECTRODE


Basic Principle:
The standard used for calibrating the dissolved oxygen electrode is the known concentration of
oxygen in air-saturated deionized water. The amount of oxygen that is dissolved in water is known
as its solubility (S) and it is dependent upon the temperature, oxygen pressure in the air, and the
concentration of dissolved solutes in the water.

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BIOL 224: Metabolism 84

Calibration Procedure:
1). Remove the oxygen electrode from the deionized storage water and place and hold the oxygen
electrode in a 100 ml beaker containing aerated deionized water (100% 02) at room
temperature, make sure the electrode does not touch the bottom of the beaker. There needs
to be enough water in the beaker to submerge the tip of the oxygen electrode.
2). Type Saturation-DI water in the Mark box to the right of Mark button on LabScribe.
3). Click Record on the Main Window. Wait until the recording reach a stable level near the top of
the recording channel. Press the Enter key on the keyboard to mark the recording when the
output of the electrode is constant. At this point in the recording, the output of the oxygen
electrode is equal to the saturation concentration of oxygen in deionized water at room
temperature. Do NOT stop recording.
4). Quickly remove the oxygen electrode from the beaker containing deionized water (100% 02).
Place and hold the oxygen electrode in a 100 ml beaker containing 0% oxygen calibration
solution (club soda / no O2) at temperature. Make sure there is enough solution in the
beaker to submerge the tip of the oxygen electrode. Make sure the electrode does not rest
on the bottom of the beaker.
5). Type No Oxygen in the Mark box to the right of the Mark button on LabScribe.
6). Click Record on the Main window. You should see a change in the recording compared to the
deionized 100% oxygen saturated water. Wait until the recording reach a stable level near
the bottom of the recording channel. Press the Enter key on the keyboard to mark the
recording when the output of the electrode is constant. At this point in the recording, the
output of the oxygen electrode is equal to no oxygen being dissolved in deionized water at
room temperature. Click Stop to halt the recording.
7). Select Save As in the File Menu. Name the file calibration.iwxdata and save it to your V:
drive or desktop.
8). Remove the electrode from the beaker of calibration solution. Place the electrode back in the
beaker containing the deionized storage water.
Unit Conversion:
1). Ask your lab demonstrator for the room temperature (in 0C). Assume the barometric pressure in
the lab room is one atmosphere (760 mmHg) and the concentration of oxygen in the air is
21%.
2). From Table 1 given below, find the dissolved oxygen concentration ([O2]) in deionized water at
room temperature. This concentration will be used in Step 6 to calibrate the dissolved
oxygen electrode.
3). Scroll to the beginning of the calibration data for the ISE-730 dissolved oxygen electrode.
4). Click the Double Cursor icon. Place one cursor on the flat section of data collected when the
saturation of dissolved oxygen in water was 100% and the second cursor on the flat section
of data collected when the saturation of dissolved oxygen in water was 0%.

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BIOL 224: Metabolism 85

Table 1. Concentration of oxygen [O2] in air-saturated deionized water at 760 mmHg.

5). Select Units from the channel menu and Simple from the Units submenu. The Simple
Units submenu will appear (Figure 34). Select 2-point calibration from the pull down menu
in the upper-left corner of the window.

Figure 3. Units conversion dialog


box in LabScribe. Note that your
values may differ from those
shown.

6). Check the Apply units to new data and Apply units to all blocks boxes.
7). Notice that the voltages from the positions of the cursor are automatically entered into the left-
hand cursor value boxes.
8). From Table 1, find the concentration of dissolved oxygen in water at the room temperature that
is 100% saturated. Enter this concentration in the corresponding box to the right of the
voltage at 100% oxygen saturation. Enter zero in the corresponding box to the right of the
voltage for 0% oxygen saturation.
9). Enter microMolar in the Unit name box. Click OK. The units conversion feature of LabScribe is
now activated and all future recordings will be automatically displayed as microMolar.

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BIOL 224: Metabolism 86

Collecting Experimental Data


Safety Issue: You will be removing solution from large carboys. The carboy hoses are double
clamped to prevent leakage of solutions. When you have obtained your solutions,
make sure the hoses are double clamped. If you do not, be prepared to mop up any spills.
1). Half-fill the 125 mL or 250 ml Erlenmeyer flask with fresh, aerated dechlorinated tap water
(Happy Fish Water).
2). Weigh the half-filled flask on the top-loading balance.
3). Ask your lab demonstrator to transfer a fish to the flask. Weigh the flask again.
4). Subtract the two weights of the flask. The difference is the weight of the fish. Enter the fish
weight (in grams) in Table 2.
5). Fill the flask to the top with fresh, aerated dechlorinated tap water. Cover the sides of the flask
with aluminum foil to minimize outside disturbance. Let the fish settle down in the flask for
about 10 minutes.
6). Make sure that the flask is full to the brim with dechlorinated tap water (there should not be any
head space). Tightly seal the mouth of flask with the rubber stopper containing the oxygen
electrode. The electrode should be at a position where the tip of the electrode is submerged
at least about inch into the water in the flask. Note that the rubber stopper should be able to
keep the electrode in place. Remember that the measurement of oxygen consumption has
to be conducted in a closed-to-air system. Make sure that there are no air bubbles inside the
flask.
7). Type Baseline Oxygen Consumption in the Mark box to the right of the Mark button.
8). Click Record and press the Enter key on the keyboard to mark the recording. Record the
output of the oxygen electrode for 30 minutes.
9). After 30 minutes, click Stop to halt the recording.
10). Select Save in the File menu.
11). Remove the rubber stopper and dissolved oxygen electrode from the flask. Place the electrode
in a beaker of deionized water.
12). Carefully pour all the water from the flask into a graduated cylinder through a dip net to catch
the fish. Return the fish to the aquarium assigned for holding fish that have been used in the
experiment. Measure the volume of water in the cylinder and record it in Table 2.
Data Analysis & Presentation
1). Use the Analysis window and the cursors to select a two-minute section near the beginning of
the recording where the slope is consistent.
2). Look at the Function Table that is above the [Oxygen] channel in the Analysis window. The
mathematical functions, V2-V1 and T2-T1, should appear in this table. The values of these
parameters are displayed in the table across the top margin of the [Oxygen] channel.

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BIOL 224: Metabolism 87

3). Record the values for the change in the oxygen concentration (V2-V1) over a two minute period
(T2-T1) in the Journal.
4). Calculate the oxygen consumption rate (micromoles/liter/minute) by dividing the value for V2-
V1, expressed as micromoles/liter, by the value for T2-T1, expressed in minutes. Enter the
value for the rate of oxygen consumption in Table 1.
5). Repeat the above steps to calculate the oxygen consumption rate (micromoles/liter/minute) from
the middle and the end section of the recording. Again, enter the values in Table 1.
6). Calculate the mean rate of oxygen consumption by averaging the rates from the beginning,
middle, and end of the recording. Enter the average value in Table 1.
7). Calculate the amount of oxygen consumed per minute (micromoles/minute) by each fish by
multiplying its mean rate of oxygen consumption (micromoles/liter/minute) by the volume
(liter) of water in the flask. Enter the value of your fish in Table 2.
8). Provide the following data to your lab demonstrator: Mean rate of 02 consumption, Volume of
water in the flask, and weight of fish. Once class data has been collected, the data will be
provided.
9). Use the data provided to create a XY scatter graph in Excel to analyze the relationship
between the oxygen consumed/minute (Y-axis) and fish weight (X-axis). Add a linear
trendline and the associated equation/value.
To add a linear trendline, click on the Layout Sub-menu. In the Analysis grouping is an icon
called Trendline, click on this and select More Trendline Options. Select Linear and also on
both Display Equation on Chart as well as Display R-squared Value on Chart. The R2 value
indicates how well the regression line fits a set of data with a value of 1.0 being a very good
fit.
10). Calculate the amount of oxygen consumed per minute per gram body weight
(micromoles/minute/gram) by each fish by dividing the oxygen consumed per minute
(micromoles/minute) by each fish by its own body weight (grams). Enter the value for each
fish in Table 2.
11). Use the data provided to create an XY scatter graph in Excel to analyze the relationship
between the oxygen consumed/minute/gram by the fish (Y axis) and fish body weight (X
axis). Add a linear trendline and the associated equation/value.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Metabolism 88

Group Assignments Due Today:


1). A figure illustrating the relationship between the oxygen consumed/minute and fish weight.
2). A figure illustrating the relationship between the oxygen consumed/minute/gram by the fish
and fish body weight.
3). A copy of the Group Data Tables from page 8.

Copy and paste the figures into a MS Word document and save as a single PDF. Be sure to
include your group number, lab day, all names (first and last) and Student ID# from your lab
group members on the first assignment page.
Hand in using Blackboard and name the file accordingly:
Group##_Lab##_LastName1LastName2LastName3.

Question to ponder: During the lab we determined that smaller fish have an increased
mass specific metabolic rate. What could be the possible physiological and ecological
consequences in small animals for having a higher than average mass-specific BMR?

Readings from the Scientific Literature: Through the link below or on reserve in the natural sciences
library.
Chaui-Berlinck J.G., Navas C.A., Monteiro, L.H.A., & J.E.P.Wilken Bicudo (2005). Control of
metabolic rate is a hidden variable in the allometric scaling of homeotherms. Journal of
Exp. Biol. 208:1709-1716 Click here for download
Gillooly, J.F., Brown, J.H., West, G.B, Savage, V.M., & E.L. Charnov (2001) Effects of Size and
Temperature on Metabolic Rate. Science, 239: 2248-2252 Click here for download
Moran, D. and R.M.G. Wells, 2007. Ontogenetic scaling of fish metabolism in the mouse-to-
elephant mass magnitude range. Comparative Biochemistry and Physiology, Part A.
148: 611-620 Click here for download
Singer, D. 2006. Size relationship of metabolic rate: Oxygen availability as the “missing link”
between structure and function? Thermochimica Acta, Volume 446, pages 20-28
Click here for download

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BIOL 224: Metabolism 89

BIOL 224 Lab Exercise #8 – Group Data Tables

Group Members: ___________________________________________

Table 1. Rate of Oxygen Consumption during different time periods of the recording.

V2-V1 [02] Change T2-T1 Rate of 02


(µmoles/liter) Time Period (min) Consumption
(µmoles/liter/min)
Beginning
Middle
End
Mean

Table 2. Rate of Oxygen Consumption of fish.

Fish Mean Rate of 02 Volume of 02 Consumed Weight 02 Consumed per


Consumption Water in per Minute of Fish Minute per Gram Body
(µmoles/liter/min) Flask (liters) (µmoles/min) (grams) Weight
(µmoles/min/gram)
1

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BIOL 224: Appendices 90

APPENDIX I: Rubric

CATEGORY Excellent Poor


FIGURE
Variables The independent variable is Axis labels and / or units are
plotted on the X-axis and the reversed.
dependent variable is plotted
on the Y-axis.

X and Y axes X and Y axes are clearly Axes are not labeled. Incorrect
labeled. Treatment groups are or no units given. Treatment
clearly labeled. Units are given groups not labeled.
in parenthesis.

Neatness Figure is professional looking, Axis range not adjusted (if


well designed, has appropriate needed) causing excess space.
axis range, appropriate spacing Overlap of axis label and axis.
between labels and axis. Figure is small and difficult to
read.

CATEGORY Excellent Good Poor Unacceptable


FIGURE LEGEND
Graph type Graph type Graph type given Graph type given Graph type not
provided and in own sentence. as an incomplete given or incorrect.
incorporated into a sentence.
sentence of
relationship
between variables

Subject Experimental Subject given in Subject is too Subject not


subject given and stand-alone general (ex. A provided.
incorporated into sentence. nerve).
another sentence
allowing for flow of
writing.
Data obtained / Clear and concise Methods of Multiple sentences No mention of
experimental set- sentence collection are outlining methods method of data
up pertaining to provided but of collection or collection.
methods of unscientific terms omission of a key
collection. are used. detail.

Relationship of Relationship Relationship Relationship Relationship


variables between variables between variables between variables between variables
clearly stated in is provided in a is difficult to not given.
first sentence of stand-alone discern / provided
legend. sentence. across two or more
sentences.

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BIOL 224: Appendices 91

CATEGORY Excellent Good Poor Unacceptable


Trend of data Trend based on Trend based on Trend of data is Trend not
graph is clearly and graph is provided difficult to discern. provided.
accurately stated. but across two or Or is provided
more sentences. twice (ex. Direct
relationship AND
as variable 1
increased variable
2 increased).
Writing style Writing is clear, Writing is concise, Writing is wordy Writing is poor
concise has good well organized and choppy. Figure with many spelling
flow, is in past with good flow, legend focus is on and grammatical
tense, well but there are a few non-essential errors. Key pieces
organized, minor omissions. aspects. Switching of information
professionally / between tenses. missing.
scientifically Writing is Disorganized.
worded. unprofessional and
unscientific.

CATEGORY Excellent Good Poor Unacceptable


CONCLUSION
Organization Information is Information is Information is Information is
well laid out with presented and somewhat disorganized and
logical flow / well organized, organized, flow is unclear.
chain of but flow of rambling.
reasoning. thought may be
wander.
Interpretation Clearly and Clearly presents Conclusions are Illogical conclusions
concisely presents key components vague. made based on data
key components. but could be Repetition of presented.
Provides an more concise. figure legend Physiological
accurate Accurate explanation not
explanation of explanation of provided.
results (or results. Flow
expected results). could be
Uses key terms improved.
correctly.
Writing style Sentences are Sentences are Sentences are Sentences are
clear and concise. mostly clear, mostly clear. fragmented or too
Tone is could be more Wording is wordy.
consistently concise. Use of a colloquial. Unprofessional and
professional and colloquial word. unscientific writing
scientific wording style.
is used.
Spelling and No spelling errors. No spelling No more than Two or more
Grammar No grammatical errors. Minor two spelling, spelling, grammatical
errors. grammatical grammatical and and / or punctuation
error. / or punctuation errors.
error.

Copyright 2019- Tracy Marchant & Sheri Fisher


BIOL 224: Appendices 92

APPENDIX II
Below is a sample figure, figure legend and conclusion that you can use as a reference.

100

90
Percent of Initial pre-cool pulse

80

70
amplitude (%)

60

50

40

30

20

10
1 2 3 4 5 6 7 8
Skinfold Measurement (mm)

Figure 1. An XY scatter graph illustrating the relationship between percent of initial of Pre-cool pulse
amplitude and the skin-fold measurement from human volunteers. As the skin fold measurement
increased, the percent of initial of pre-cool amplitude increased. The further away from 100% Pre-
cool, the larger the change in pulse amplitude during the Cool treatment.

Conclusion:
Skin fold measurement is indicative of subcutaneous adipose tissue, which acts as an insulating
layer. Insulation not only affects the time it takes for thermoreceptors to detect a change in
temperature and initiate an appropriate response but also helps to retain heat. Therefore, the pulse
amplitude of subjects with larger skin-fold measurements was not as affected by the ice treatment
as subjects with lower skin-fold measurements allowing their pulse amplitude to remain closer to
the Pre-cool measurement.

Copyright 2019- Tracy Marchant & Sheri Fisher