University of Warwick
Headstart Summer
School
Physiology Lab
Practical manual
Testing Breathing and Heart Rate
In the first part of this laboratory session, you will be introduced to spirometry as a
technique for recording respiration.
In the second part, you will be introduced to ECG as a method of measuring heart
rate.
If we have time in groups you will come up with your own hypothesis for which you
will design and experiment using one or both of these techniques.
Part 1: Respiratory Airflow and Volume
Background
Gas exchange between air and blood occurs in the alveolar air sacs. The efficiency of
gas exchange is dependent on ventilation. Cyclical breathing movements alternately
inflate and deflate the alveolar air sacs (Figure 1). Inspiration provides the alveoli
with some fresh atmospheric air, and expiration removes some of the stale air,
which has reduced oxygen and increased carbon dioxide concentrations.
Figure 1: Basic Lung Anatomy
Spirometry is becoming more important as respiratory diseases are increasing
worldwide. Spirometry is the method of choice for a fast and reliable screening of
patients suspected of having Chronic Obstructive Pulmonary Disease (COPD). COPD
is the 12th leading cause of death worldwide and the 5th leading cause in Western
countries. Studies suggest COPD could climb to be the 3rd leading killer by 2020.
Most COPD cases are completely avoidable as 85-90% of cases are caused by
tobacco smoking.
Many important aspects of lung function can be determined by measuring airflow
and the corresponding changes in lung volume. Airflow can be measured directly
with a pneumotachometer (from Greek roots meaning “breath speed measuring
device”). The PowerLab pneumotachometer is shown in Figure 2.
Figure 2: The PowerLab pneumotachometer.
The flow head measures the difference in pressure either side of a mesh membrane
with known resistance. This resistance gives rise to a small pressure difference
proportional to flow rate. Two small plastic tubes transmit this pressure difference
to the Spirometer Pod, where a transducer converts the pressure signal into a
changing voltage that is recorded by the PowerLab and displayed in LabChart. The
volume, V, is then calculated as the integral of flow:
V=

 F dt
This integration represents a summation over time. Sometimes the measurements
drift due to differences in temperature and humidity between inspiration and
expiration. Computer software corrections used in the Spirometry Extension can
reduce drift but cannot eliminate it.
Spirometry allows many components of pulmonary function to be visualized,
measured, and calculated (Figure 3). Respiration consists of repeated cycles of
inspiration followed by expiration. During the respiratory cycle, a volume of air is
drawn into and then expired from the lungs. This volume is the tidal volume (V T). In
normal ventilation, the breathing frequency (ƒ) is approximately 15 respiratory cycles
per minute. This value varies with the level of activity. The product of ƒ and VT is the
expired minute volume (VE), the amount of air exhaled in one minute of breathing.
This parameter also changes according to the level of activity.
Figure 3: Lung volumes and capacities.
Other parameters include Inspiratory and Expiratory Reserve Volumes (IRV and ERV),
which is the volume of extra air you inspire or expire when breathing in and out as
hard as possible.
The Vital Capacity (VC) is the total volume is the total amount of air that can be
expired after fully inhaling.
Even when a person exhales completely, there is still air left in the lungs. This
leftover air is the Residual Volume (RV), which cannot be measured by spirometry.
The Total Lung Capacity (TLC) is the sum of the Vital Capacity and Residual Volume
(VC + RV).
Term
Breathing Frequency
Expired Minute Volume
Lung Volumes
Tidal Volume
Inspiratory Reserve Volume
Expiratory Reserve Volume
Residual Volume
Lung Capacities
Inspiratory Capacity
Expiratory Capacity
Vital Capacity
Total Lung Capacity
Abbreviation / Symbol
f
VE = f x V T
Units
Resp. cycles/min
L/min
VT
IRV
ERV
RV (predicted)
L
L
L
L
IC = VT + IRV
EC = VT + ERV
VC = IRV + ERV + VT
TLC = VC + RV
L
L
L
L
Table 1: Summary of Spirometry terms
4. Procedures
If you are suffering from a respiratory infection, do not volunteer for this
experiment.
A. Equipment Setup
Make sure the PowerLab is turned off and the USB cable is connected to the
computer.
Connect the Spirometer Pod to Input 1 on the front panel of the PowerLab (Figure
4). Turn on the PowerLab.
Note: Since the Spirometer Pod is sensitive to temperature and tends to drift during
warm-up, it is recommended the PowerLab (and therefore the Spirometer Pod) is
turned on for at least 10 minutes before use. To prevent temperature drift, place
the Spirometer Pod in a shelf or beside the PowerLab, away from the PowerLab
power supply to avoid heating.
Figure 4: Equipment Setup for PowerLab 26T
Connect the two plastic tubes from the Respiratory Flow Head to the short pipes on
the back of the Spirometer Pod. Attach Clean-bore Tubing, a Filter, and a
Mouthpiece to the Flow Head (Figure 4).
B. Hygiene
NOTE:
Each volunteer should use a clean mouthpiece and air filter.
The vinyl mouthpiece should be cleaned between uses by soaking it disinfectant
provided.
A new air filter will be supplied for each volunteer.
# 1: Familiarize Yourself with the Equipment
In this exercise, you will learn the principles of spirometry and how integration of the
flow signal gives a volume. The equipment should already be calibrated, the next
section is for reference, and you should start at ‘Using the Equipment’.
Calibrating the Spirometer Pod – we will set this for you – but if you accidentally power down the
computer of stop the programme this will need to be reset
The Spirometer Pod must be calibrated before starting this exercise. The Flow Head must be left
undisturbed on the table during the zeroing process.
1.
Launch LabChart and open the settings file in “Respiratory Airflow & Volume” from the
Experiments tab in the Welcome Center. It will be located in the folder for this experiment.
Select Spirometer Pod from the Channel 1 Channel Function pop-up menu. Make sure the Range is
500 mV and the Low Pass is 10 Hz; then select Zero. When the value remains at 0.0 mV, have the
volunteer breathe out gently through the Flow Head, and observe the signal (Figure 5). If the signal
shows a downward deflection (it is negative), you can return to the Chart View. If the signal deflects
upward, you need to invert it. Click the Invert checkbox once.
Figure 5: Spirometer Pod Dialog with Downward Deflection
Note: The signal can also be inverted by reversing the orientation of the Flow Head or by swapping
the connections to the Spirometer Pod. The Invert checkbox is more convenient.
Using the Equipment
2. Have the volunteer put the Mouthpiece in their mouth and hold the Flow Head
carefully with both hands. The two plastic tubes should be pointing upward.
Put the nose clip on the volunteer’s nose. This ensures that all air breathed passes
through the Mouthpiece, Filter, and Flow Head (Figure 6).
Figure 6: Proper Positioning of the Flow Head
After the volunteer becomes accustomed to the apparatus and begins breathing
normally, you are ready to begin.
Start recording. Have the volunteer breathe normally and record the volunteer’s
tidal breathing for one minute. At the end of one minute, have the volunteer
perform a full expiration. Observe the data being recorded in the “Flow” channel.
Stop recording.
The volunteer can stop breathing through the Flow Head and can remove the Nose
Clip.
Setting Up the Spirometry Extension: This is for reference
The Spirometry Extension processes the raw voltage signal from the Spirometer Pod, applies a volume
correction factor to improve accuracy, and displays calibrated Flow (L/s) and Volume (L) traces. It
takes over from Units Conversion. The trace you recorded in this exercise will provide reference
points for the Spirometry Extension that allow it to calculate and perform corrections on the trace.
3. Drag across the Time axis at the bottom of the Chart
View to select the data you recorded. Select Spirometry
Flow from the Channel 1 Channel Function pop-up menu.
Make sure the settings are the same as those in Figure 7.
Select Spirometry Volume from the Channel 2 Channel
Function pop-up menu. Make sure Channel 1 is selected in
the Spirometry Flow Data pop-up menu. Click the Apply
Volume Correction checkbox to turn it on. Then select
Apply to allow the extension to use the volume correction
ratio that is has calculated from your data (Figure 8). The
Chart View should now appear with calculated volume data
on Channel 2. This can be problematic if the initial tidal
breathing recorded is erratic.
Figure 7: Spirometry Flow
Dialog
Figure 8: Spirometry Volume Dialog
Select Set Scale from the Scale pop-up menu in the Amplitude axis for the “Flow” channel. Make the
top value 15 L/s and the bottom value -15 L/s.
Understanding the Importance of Volume Correction:
4. Drag across the Time axis to select data from both channels, and open Zoom
View. Note the relation between Flow and Volume. When the flow signal is
positive (inspiration), “Volume” rises and when the flow is negative (expiration),
the volume trace falls.
5. In Zoom View, find part of the recording where the flow is zero. Note that at this
time, “Volume” does not change (it is horizontal) because integrating a zero
signal does not add anything to the integral.
6. The volume trace is calculated by the extension in such a way that the displayed
volumes at the end of the two full expirations equal. In subsequent recordings,
the volume correction is unlikely to be exact – you will notice a tendency for the
volume to drift, typically 1-2 L over 1-2 minutes. To see the effect of having no
correction, turn off the Volume Correction checkbox in the Spirometry Volume
dialog, and examine the data trace. Remember to turn it back on again
afterward.
7. Do not close the file.
#2: Spirometry Recording
From this recoding, you will examine the respiratory cycle, measure changes in
flow and volume - 1) Lung volume capacities; and measure parameters of forced
expiration that are used in evaluating 2) Pulmonary function. Note that the
Spirometry Extension is not intended for clinical evaluation of lung function.
1. Zero the Spirometer Pod again, using the same procedure as before.
Remember to leave the Flow Head undisturbed during the process.
2. Have the volunteer face away from the monitor and relax, get them to read
something. This will prevent the volunteer from consciously controlling their
breathing during the exercise.
3. When ready, Start recording. After two seconds, have the volunteer replace
the Nose Clip and breathe normally into the Flow Head. Record normal tidal
breathing for one minute (remember to add a comment with “normal tidal
breathing” to the data trace).
4. After the tidal breathing period (at the end of a normal tidal expiration), ask
the volunteer to inhale as deeply as possible and then exhale as deeply as
possible. The volunteer should focus on depth of breathing not speed
(remember to add a comment of “lung volume procedure” at this position).
Afterwards, allow the volunteer to return to normal, relaxed, tidal breathing
for approximately 30 seconds.
5. When ready, ask the volunteer to inhale maximally and then exhale as
rapidly, forcefully and fully as possible (that is, inhale as much as possible
and then exhale until no more air can be expired). The volunteer must this
time focus on depth and speed. The volunteer should let his or her breathing
return to normal then repeat this procedure twice more (remember to add a
comment “forced breathing 1”, “Forced breathing 2” and “forced breathing
3”)
Figure 9: Sample Data of Forced Breaths
6. Save your data. Do not close the file.
7. You may need to repeat this entire procedure to ensure the data are valid.
8. N.B. On-screen data traces can be copied by performing a screen capture
(press Control and print screen keys simultaneously, then paste the copy into
a suitable application, e.g. Word, PowerPoint or WordPad, and save the
document).
In the next section measure and record key spirometry values
3.1 Lung Volumes and Capacities (Record in Table 2, p13)
1. Examine the normal tidal breathing data. Calculate how many respiratory
cycles there are in a one minute period. This is the breathing frequency (f); it has
units of respiratory cycles per minute (respiratory cycles/min). Record this value
in Table 2.
2. Determine the volume of a single tidal inspiration by placing the Marker
(bottom left of screen) at the start of a normal tidal inspiration. Place the
Waveform Cursor at the peak (Figure 10). This will be about0.5-1.5 s to the
right of the marker. The value shown in the Range/Amplitude display for
Channel 2 is the tidal volume (VT) for that breath. Record this value in Table 2.
Figure 10: Proper Placement of Marker and Waveform Cursor
3. Return the Marker to its box at bottom left of the Chart View, by doubleclicking the Marker, dragging it back, or clicking its box.
4. Using the value for VT and the number of respiratory cycles observed over a
one-minute period (f), calculate the minute volume (VE) using Equation 3. Record
your value in Table 2.
VE = f x V T
(L/min)
Equation 3
5. Find the “Lung volume procedure” comment in your data trace. Repeat steps
2-3 to determine the inspiratory reserve volume (IRV) (Figure 11) and expiratory
reserve volume (ERV) (Figure 12). Record these values in Table 2.
Note: the Marker should remain at the start of a normal tidal inspiration (trough)
for the ERV, and it should be moved to the end of a normal tidal inspiration
(peak) for the IRV. If necessary, refer to Figure 3 for further clarification.
Figure 11: Positioning of Marker and Waveform Cursor to Measure IRV
Figure 12: Positioning of Marker and Waveform Cursor to Measure ERV
Respiratory
parameter
Breathing
frequency
Expired Minute
Volume
Tidal Volume
Inspiratory
Reserve Volume
Inspiratory
Capacity
Expiratory
Reserve Volume
Expiratory
Capacity
Vital Capacity
Residual Volume
Total Lung
Capacity
Abbreviation
Units
f
Resp.
cycles/min
VE = f x V T
L/min
VT
L
IRV
L
IC= VT + IRV
L
ERV
L
EC= VT + ERV
L
VC (from table)
VC = IRV + ERV
+ VT
RV = pred. VC x
0.25
L
TLC = VC + RV
L
Experimental &
calculated
value
L
L
Table 2: Spirometry measurements
Spirometry
Extension
value
Part 2. The Electrocardiogram (ECG)
In this practical, you will use electrodes placed on the skin of the wrists and ankles to
record the ECG from members of your practical group and will relate this recording
to the pulse, recorded using a pulse transducer placed on a finger, and to the heart
sounds, recorded using a microphone placed on the upper part of the chest.
The ECG. The heart beat is initiated in the sinoatrial node and is conducted by
electrical means through the heart in such a way that contraction ejects blood
efficiently into the aorta (from the left ventricle) and the pulmonary trunk (from the
right ventricle). The electrical means of conduction is associated with the flow of
current in extracellular fluid, and the effects of this current can be picked up by
electrodes placed at appropriate positions on the skin. The ECG can be represented
diagrammatically as a graph of recorded voltage against time:
QRS
P
T
Figure 1 Idealised ECG, indicating the principal waveforms and the intervals used clinically to assess
normal or abnormal conduction of the heart beat.
The general form may be familiar to you. The P wave denotes the start of atrial
depolarisation; the QRS complex the onset of ventricular depolarisation; and the T
wave the repolarisation of the ventricle. Information about diseases of the heart can
be obtained clinically from changes in this basic pattern.
Since the heart beat is conducted through a complex, three dimensional structure,
clinicians use several ‘leads’ to look at its spread from several different aspects.
Electrodes attached to the limbs and to the chest are used. You will look at three of
the limb leads.
Recording the ECG.
You will record the ECG using so-called bipolar limb leads. These leads are denoted
I, II, and III by clinicians.
If at any stage you are unsure, consult a demonstrator.
Attaching the recording electrodes
1. With the PowerLab turned off, plug the Bio Amp cable into the Bio Amp.
2. The volunteer should remove his/her watch and any jewellery from his/her
wrists.
3. Connect the lead wires to earth, CH1 negative, CH1 positive on the Bio Amp
cable.
4. Stick one disposable electrode onto your left wrist and two electrodes onto
your right wrist.
5. Connect the:
Right wrist to earth
Right wrist to CH1 negative
Left wrist to CH1 positive
These connections will give recordings equivalent to lead I used by a
cardiologist (see Fig. 3).
6. Ensure that the volunteer is comfortable and is able to sit still and relaxed
during the period of recording (a few s). Activity in skeletal muscle will
interfere with the recording, so the quality of your recording will be affected
by this ability of the subject to relax. The subject may find it best to sit with
his/her hands resting lightly one on each knee. In a clinical recording, the
subject would lie flat on a bed.
Software set up – the demonstrators will do this for you – but is here for information
1. Turn on the PowerLab
2. Launch Chart7 from the computer.
3. Set up Chart7 so that you can record from Channel 3 (the channel to which
the BioAmp lead connects).
Click on Setup, then Channel settings. Make sure channels 3 and 4 are on.
Set the number in the Range column to 2mV in each case. Set n at the
bottom left of the panel to 4. OK these settings to return to the recording
chart.
4. Set the digitisation rate (top right of the screen) to 1k/s.
Recording the ECG.
Objective: To measure the ECG, identify the major elements and measure the heart
rate and other (PR, ST, QT) intervals (Fig. 1).
Procedure
1. Click the Start button and record for approximately 10s. You will be
recording lead I on Channel 3 and lead II on Channel 4. Make sure you get
some nice clean data: repeat if noisy.
2. Click the Stop button.
3. Save the results by choosing Save as . . . . from the File menu in Chart.
4. Alter the connections of the electrodes so that the left wrist (rather than the
right wrist) is connected to CH2 negative. The other connections should
remain as before (right ankle to earth, left ankle to CH2 positive, left wrist
still connected also to CH1 positive, right wrist still connected to CH1
negative). On Channel 4 you will now be recording using lead III in the
nomenclature used by the cardiologist (see Fig. 3), but will continue to record
lead I on Channel 3. Repeat steps 1 – 2.
5. Save the results as in step 3.
If you cannot see the ECG, check that the electrodes are correctly attached and that
the sensitivity range of the amplifier is sufficient. Adjust the ‘gain’ of the recording if
necessary. If the recording is noisy, ensure that the volunteer is relaxed, comfortable,
and sitting still. If you still have trouble, remove the connections to CH2 of the Bio
Amp cable so that you are recording only lead I.
Analysis
Timing of the heart beat. Examine the ECGs you have recorded and identify the P
wave, the QRS complex, and the T wave.
Measure the heart rate as follows. Put the marker cursor (M) at the top of one R
wave and move the cursor on several beats to the top of another R wave and note
t
t in s) and number of
beats (n) to work out the heart rate in beats per minute. This will be given by:
rate 
n 1
 60 beats.min-1
t
In one of the recordings (lead II, say), measure the other intervals or durations of
wave forms in the ECG indicated in Figure 1 and in the Table.
Table 1 Resting ECG
Subject
Heart rate
RR (s)
(beats.min-1)
PR (s)
QRS
ST (s)
QT (s)
duration (s)
Can you say from your results whether systole or diastole lasts longer in resting
conditions?
If the P wave was not followed by a QRS what could this signify?
What could a long QRS duration signify?
What is long QT syndrome?
At the end of this manual, there are additional notes on ECG and how to take further
measurements
Part 3: Experimental Design – TIME PERMITTING!
In groups of 4, you will come up with your own hypothesis for which you will design
and experiment using one or both of these techniques. (You will be unable to use
the techniques simultaneously on 1 person.)
Some suggested experiment titles





The effect of exercise on heart rate/breathing
The effects of holding breath and hyperventilation on ECG
The effect of meditation on the heart rate
Does posture have an effect on breathing?
“The effect of caffeine on heart rate and/or breathing”
When you are thinking of what experiment you want to do, consider what conditions
you will compare. You may want to compare duration, intensity or type of exercise,
or look at individual responses to the same exercise comparing gender, age or fitness
level.
Note that individual must be disconnected from ECG whilst exercising and quickly
reattached once finished.
Before you start, you will need to:
 Write a hypothesis
 Summarise your experimental methods
 Work out what results you want to measure and how best to record them
 Prepare a table for results to be recorded
Remember, “The difference between screwing around and science is writing it
down”. (Adam Savage, Mythbusters)
A note about safety: Before starting any experiment, please check with a lab
demonstrator that they are happy that it is safe!
Experiment Title: _______________________________________________________
Hypothesis:
Experimental Design:
Results:
Further exercises: The ECG and the heart sounds.
Heart sounds. Heart sounds are associated with the shutting of heart valves. Two
distinct sounds are normally present. The first, duller sound (often denoted lub) is
associated with the shutting of the mitral and tricupsid valves in the left and right
sides of the heart, respectively. The second, sharper sound (dub) is associated with
shutting of the aortic and pulmonary valves. Even in normal physiological
conditions, this second heart sound may be split owing to the aortic and pulmonary
valves shutting at slightly different times. Such splitting is normally more prominent
during inspiration, when the pulmonary valve shuts slightly later than the aortic.
(Why do you think splitting of the second heart sound happens in this way?)
Errors of normal heart valve opening or closure generally result in blood flowing
turbulently through valves, and this turbulence can be detected as a murmur. A
third heart sound can often be detected and is normal, especially in young people. It
is associated with rapid, turbulent flow of blood into the ventricles from the atria at
the start of diastole.
Heart sounds are detected by auscultation using a stethoscope or with a microphone
placed in an appropriate position, and you will listen to your own heart sounds in
this way during this practical class. Physicians listen in turn at each of four distinct
sites on the chest for heart sounds. These positions optimise detection of sounds
from particular valves and are shown in Figure 2. In this practical, you will pick up
heart sounds from the aortic area.
aortic area
pulmonary
area
tricuspid area
mitral area
Figure 2. Positions used in auscultation. The aortic and pulmonary areas are at the sternal edge at
the level of the second intercostal space on right and left sides, respectively. The tricuspid area is at
the left sternal edge at the level of the fifth intercostal space; the mitral area is at the level of the fifth
intercostal space in the mid clavicular line.
In this exercise, you will record the heart sounds from the aortic area, over the
second intercostal space at the right edge of the sternum (see Fig. 2). This is the
place where the ascending aorta comes closest to the chest wall.
First, each member of the group should listen to their own heart sounds using the
stethoscope. Place the bell of the stethoscope on the chest under your shirt or top
and search for a place where you can readily hear your heart sounds. Identify the
first and second of these. Examine the effect of breathing in and holding your breath
for a brief period. Can you hear splitting of the second heart sound (this is difficult)?
Finding the second intercostal space. First find the clavicle or collar bone. Run your
finger along it from lateral to medial until you come to the place where it meets the
sternum at the manubrium sterni. The first rib also attaches just below the clavicle.
Identify the first intercostal space, then the second rib, and then the second
intercostal space in order below the sternal end of the clavicle.
Connect the volunteer as follows:
1. Place the microphone over the second intercostal space at the right sternal
edge.
2. Tape the microphone into position here, using the tape provided.
3. Reconnect the ECG electrodes to record whichever lead (I, II, or III) gives the
largest QRS complex.
Software set up
1. Set up Chart so that you can record from Channels 1 and 3. You will use
Channel 3 to record the ECG as before. You will use Channel 1 to record the
heart sounds.
2. Click on Setup, then on Channel Settings. Make sure channels 1 and 3 are on.
Set the number in the Range column to 20mV for Channel 1, and 2mV for
Channel 3. Set n to 3.
3. In the Input Amplifier column, click on Input Amplifier for Channel 1. In the
menu that then appears, make sure the Channel is recording Input 1. In the
Calculation column, make sure the setting for Channel 1 is on No Calculation.
4. OK these settings to return to the recording chart.
5. Set the digitisation rate to 1k/s.
6. Record for about 10s.
7. The heart sounds are difficult to record. You may need to move the
microphone slightly to ensure it picks them up. Sometimes filtering the record
can help
Analysis
Identify the waveforms of the ECG. Identify the first and second heart sounds.
Use one of the several heart beats you have recorded to make the following
measurements.
1. Use the cursor to measure the time at the start of one of the QRS complexes
and at the start of the following T-wave of the ECG. Remind yourself what
events these signify?
2. Use the cursor to measure the start of the first (S1) and second (S2) heart
sounds.
3. Work out the interval between the first and second heart sounds.
4. Work out the intervals between the QRS complex and the first heart sound,
between the T wave and the second heart sound.
5. Write these intervals in the following Table.
Table 4 Relationship between the ECG and the heart sounds.
Subject
Interval S1 to S2
Interval QRS
and S1
Interval T wave
and S2
From the above table:
What is the duration of systole?
What is the duration of diastole?
Why is there a delay between the QRS complex and the first heart sound (S 1)? What
links the two?
Why is there a delay between the T wave and the second heart sound (S 2)? What
links these two events?
Electrical axis of the heart. Look at the QRS complexes in the three leads (I, II, and III,
the different modes of connecting electrodes to the amplifier). Are they identical in
each case? It is unlikely that they will be – why?
Measure the amplitude (in mV) of the R wave (relative to the baseline) with the
cursor in each of the 3 leads.
Table 2 Electrical axis of the heart
Subject
Amplitude in lead I Amplitude in Lead Amplitude in Lead
(mV)
II (mV)
III (mV)
Note that at this time the heart beat will be spreading through the ventricular
musculature. In which net direction is the heart beat spreading at this time? The Rwave is often biggest in Lead II (Fig. 3). However, there is a very large variation from
one individual to another in the electrical axis of the heart, so do not be concerned if
the electrical axis does not fit this prediction. What factors determine the axis of the
heart?
Figure 3. The connections used for recording leads I, II, and III are shown on the left. The directions in
which they are considered to ‘view’ the heart are shown on the right.