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Students are reminded that they must have an access code to view or print the lab manual.
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
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
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.
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.
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
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.
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.
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).
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.
Figure 1: The front (at top) and rear (at bottom) panels of the iWorx 214.
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.
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.
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
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.
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.
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.
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).
Mark box
Downward arrow
Channel
window
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.
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.
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.
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…
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.
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
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).
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.
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
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
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!
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
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.
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.
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.
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.
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.
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).
-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.
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.
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
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.
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.
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.
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
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).
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.
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.
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).
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.
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.
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.
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.
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.
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.
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
Important Notes: Volunteers for Experiment 1 need to wear shorts. Please bring these with you to this
lab exercise. A changing room is available.
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.
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).
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.
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.
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:
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.
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.
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.
Enter the
calibration
weight here
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.
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
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.
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.
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.
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.
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.
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.
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
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
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
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).
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.
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.
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.
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.
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
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.
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.
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.
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
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.
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.
EQUIPMENT SET-UP
1). Locate the C-AAMI-504 cable, electrode lead wires and pulse plethysmograph (Fig. 2).
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.
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.
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.
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.
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 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
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)
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.
3
10
Endotherms, 39°
-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).
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.
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 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%.
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.
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.
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.
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
Table 1. Rate of Oxygen Consumption during different time periods of the recording.
APPENDIX I: Rubric
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.
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.