TABLE OF CONTENTS

page
General Laboratory Instructions 2
Preparation of the Animals for Experimentation 3
Introduction to LabTutor 4

The Cell
Exercise I – Hemolysis and Crenation 18

The Respiratory System

Exercise II – Respiratory Air Flow and Volume 20
Exercise III – Mechanics of Ventilation 33
Exercise IV – Breathing 50

The Cardiovascular System

Exercise V – Blood Pressure 61
Exercise VI – ECG and Heart Sounds 69

The Renal & Genitourinary System

Exercise VII – Kidney and Urine 80
Exercise VIII– Water Balance 92

The Digestive System & Nutrition

Exercise IX – Gut Absorption 102

The Neuromusculoskeletal System

Exercise X– Frog Skeletal Muscle 110
Exercise XI – Frog Neuromuscular Junction 131
Exercise XII – Reflexes 146
Exercise XIII– Sensory Physiology 152

Immunohematology
Exercise XIV- Blood 176
 TABLE OF CONTENTS

page
General Laboratory Instructions 2
Preparation of the Animals for Experimentation 3
Introduction to LabTutor 4

The Cell
Exercise I – Hemolysis and Crenation 15

The Respiratory System

Exercise II – Respiratory Air Flow and Volume 16
Exercise III – Mechanics of Ventilation 24
Exercise IV – Breathing 34

The Cardiovascular System

Exercise V – Blood Pressure 40
Exercise VI – ECG and Heart Sounds 44

The Renal & Genitourinary System

Exercise VII – Kidney and Urine 51
Exercise VIII– Water Balance 57

The Digestive System & Nutrition

Exercise IX – Gut Absorption 61

The Neuromusculoskeletal System

Exercise X– Frog Skeletal Muscle 65
Exercise XI – Frog Neuromuscular Junction 76
Exercise XII – Reflexes 85
Exercise XIII– Sensory Physiology 89

Immunohematology
Exercise XIV- Blood 106
 TABLE OF CONTENTS

page
Introduction to LabTutor 2

The Cell
Exercise I – Hemolysis and Crenation 5

The Respiratory System

Exercise II – Respiratory Air Flow and Volume 6
Exercise III – Mechanics of Ventilation 11
Exercise IV – Breathing 18

The Cardiovascular System

Exercise V – Blood Pressure 23
Exercise VI – ECG and Heart Sounds 27

The Renal & Genitourinary System

Exercise VII – Kidney and Urine 31
Exercise VIII– Water Balance 37

The Digestive System & Nutrition

Exercise IX – Gut Absorption 43

The Neuromusculoskeletal System

Exercise X– Frog Skeletal Muscle 47
Exercise XI – Frog Neuromuscular Junction 57
Exercise XII – Reflexes 63
Exercise XIII– Sensory Physiology 65

Immunohematology
Exercise XIV- Blood 72
 GENERAL LABORATORY INSTRUCTIONS

1. Study the experiment well in advance.

2. For smooth teamwork, the group leader should assign each member to a particular
task or tasks. The student will be graded on teamwork as well as on individual
accomplishments.
3. Each group of students must keep a loose-leaf notebook for recording
observations and drawing data obtained from the experiment, which will be
checked at the end of the laboratory period.
4. A student will be assigned to lead the class discussion of the results obtained.
5. Provide yourself with a laboratory gown. This shall be regularly enforced.
6. Seek advice from the laboratory preceptors whenever you are unsure of anything.
7. Don’t smoke cigarettes or the like inside the laboratory.
8. Never leave your working table until it has been wiped clean with rug after each
laboratory period.
9. Attendance will be checked anytime during the laboratory period.
10. Submit your workbook for grading one day after the discussion of a particular
experiment or as announced by the instructor. Failure to submit on or before the
deadline will result in a failing grade for the particular experiment.
 PREPARATION OF THE ANIMALS FOR EXPERIMENTATION

For several experiments in which the function of the muscles and nerve tissue is
to be examined, the required tissue will be excised from the leg of the frog. The frog will
first be brain pithed. This is a procedure whereby the brain tissue is quickly destroyed
rendering the animal insensitive to pain while other tissues remain intact and functional
for a considerable time.

BRAIN PITHING PROCEDURE

The frog is held in one hand with the dorsal side up and the animal’s head
directed away from the examiner. The frog’s head is held beneath the forefinger and bent
slightly downward. The position of the foramen magnum is located by drawing a
dissecting needle slowly posteriorly along a medial line over the skull of the frog until a
slight depression at the base of the skull is felt. The needle is quickly inserted in the
foramen, pushed further into the cranium, and the needle point moved from side to side.

MUSCLE NERVE PREPARATION

Double pith the frog. Remove the skin by cutting all around the mid-region and
pulling it off completely at the toes. Lift the tip of the urostyle (the long bone found at the
tip of the spinal column) and remove it by transverse section at the level of the last
vertebra.

Lay the frog on its vertical side and using blunt dissection, tear through the fascia
between the semi-membranosus and vastus externus muscles. The sciatic nerve and
accompanying vessels lie in the sulcus between the muscles.

Using a glass probe, lift the nerve and tie a lifting ligature as it comes out from the
spinal cord. Cut the nerve proximal to the ligature and carefully trace it downward until
the region of the knee. Avoid stretching the nerve unnecessarily. Keep it moist with
Ringer’s solution.

Remove all the thigh muscles but retain the femur.

Measure the resting length of the gastrocnemius muscle in mm from its origin at
the femur to its insertion at the proximal level of the tendon of Achilles with the leg in
neutral position (semi-flexed). Set up the frog sciatic-gastrocnemius-femur for
experimentation.
 INTRODUCTION TO LabTutor

LabTutor is an HTML-based software package, designed specifically for laboratory

teaching and used in conjunction with ADInstruments' PowerLab.

LabTutor controls the sampling, digitizing and storage of experimental data, and allows
you to display, manipulate and analyze them. The technology derives from equipment
such as the Ludwig drum kymograph which recorded data on a revolving drum. This was
replaced by paper chart-recorders, such as the Grass polygraph. Then came computer-
based data acquisition systems. One of the first of these was MacLab, the predecessor of
the ADInstruments PowerLab/LabTutor system, which you are using in this course.

The principle of displaying data in the manner of a paper-chart recorder has not changed.
A LabTutor display panel is essentially a window showing a section of a conceptual strip
of electronic data chart.

1890’s
Drum Kymograph
according to
Ludwig

1960’s
Grass
Polygraph

Today, the PowerLab and

Labtutor Panel, displaying a
section of recorded data
Learning Objectives

By the end of today's laboratory you will be:

• Familiar with the major hardware and software components of the LabTutor
system
• Able to record basic finger-pulse signals in LabTutor
• Able to use some of the annotation and analysis features in the LabTutor software

Data Acquisition

First, a transducer converts the signal of interest (for example blood pressure or body
temperature) into an analog voltage, whose amplitude usually varies over time. This in
turn is monitored by the recording hardware, which can modify the signal by
amplification and filtering, processes called 'signal conditioning'. The resulting signal is
sampled at regular intervals and converted from analog to digital form before
transmission to the attached computer, where the sampled data is stored and displayed.

Data acquisition with a PowerLab system

The PowerLab Hardware Unit

The basic hardware unit is the PowerLab, a multi channel recording instrument for the
measurement of electrical signals. Many of the teaching models of PowerLabs include an
isolated stimulator for electrical stimulation of nerve and muscle, as well as integrated
two channel Bio Amps for optimal recording of biological signals.

In your experiments, you simply attach appropriate cables to connectors on the front of
the PowerLab and measure the signals in LabTutor.

Signal Conditioners

ADInstruments signal conditioners are software-controlled pre-amplifiers for use with

PowerLab data acquisition systems. The two types you are likely to see are Pods and
Front-ends. Once connected, the signal conditioners are automatically identified by the
PowerLab system, and all settings are stored when the data file is saved on your
computer.

Types of signal conditioners you may use in this course

Left to right:
Pod used for electro-oculography;
Amplifier for measuring galvanic skin resistance;
Bridge amplifier for connection to force and pressure transducers.
Signal Transducers

Virtually any transducer that generates an analog voltage between ±10V may be attached
to the PowerLab. Either directly or through one of the signal conditioners mentioned
above.

Event markers
Some transducers may be used to mark
an event, like this pushbutton switch.

Stimulation of nerves and muscles

PowerLab also has the ability to produce
an output voltage to induce a response in
a nerve or muscle. This stimulating bar
electrode connects to the Isolated
Stimulator on the PowerLab.

Force or pressure transducers

Some transducers measure force like this
hand dynamometer.

Chemical properties
Chemical properties may also be
measured such as conductivity, dissolved
oxygen, and pH.
Connecting Your PowerLab

Any number of transducers may be connected to the available inputs.

Front panel of your PowerLab

1. Power indicator light: Illuminates when PowerLab is turned on.

2. Analog output connections: provides a voltage output in the range 10 V.
NOT safe for direct connection to humans.
3. Analog inputs: Inputs on the PowerLab for connecting transducers and devices
with BNC connectors (not present on all models).
4. Isolated Stimulator status light: Indicates that the Isolated Stimulator is working
properly (green) or out of compliance (yellow).
5. Dual Bio Amp input: Connects a 5 lead Bio Amp cable to the PowerLab; reads as
Inputs 3 and 4.
Safe for direct connection to humans.
6. Isolated Stimulator outputs: For connecting stimulating electrodes to the Isolated
Stimulator.
Safe for direct connection to humans.
7. Isolated Stimulator switch: Turns the Isolated Stimulator on and off.
8. Pod ports: 8-pin connectors for attaching Pods and certain transducers to Inputs.
These also supply a DC power to the pods and transducers.
9. Trigger input, may be used to start or stop a recording event (not present on all
models).

DO NOT connect a device to the Analog Input and to the Pod port on the same
channel.
 Rear panel of your PowerLab

10. Audio output connector: Standard 1/8" (3 mm) phono jack for sound output of
recordings from the Bio Amp (not present on all models).
11. Earthing post: Used to ground PowerLab, if grounded power supply is
unavailable.
12. Power switch.
13. I2C connector: Connects PowerLab to special ADInstruments signal conditioners
called front-ends (not present on all models).
14. USB connector: for connecting a computer to the PowerLab.
15. Serial Port connector: Connects PowerLab to certain devices (not present on all
models).
16. Power cord connector.
17. Digital Input connector (not present on all models).
18. Digital Output connector (not present on all models).
Organization of LabTutor Experiments

Every LabTutor experiment is organized in basically the same way.

From the experiment index you will find a link to the introductory page of the assigned
experiment which may already be pre-loaded onto your computer.

Every experiment begins with an introductory page. On this page there is a brief
introduction and a list of learning objectives. The subsequent exercises allow you to
accomplish the specified learning objectives.

Each exercise includes highlighted text with links to pop-up windows containing
additional information, helpful tips, and useful references to LabTutor features.

Each exercise page contains a LabTutor panel in which data is recorded.

After each Exercise page, there is an Analysis page. Data that you recorded during the
exercise is available here for you to make measurements and complete any tables or
graphs that are required.

At the end of the experiment is the Report section. Any recordings that are required for
your report are reproduced here, along with tables or graphs that you have completed.
This section also contains questions that you can answer by typing into the spaces
provided. Your instructor will advise you how to submit your completed lab report.

All pages include a link in the footer to the Background material. This provides some
general information regarding the science behind the experiment. It may have been given
to you by your instructor prior to your laboratory.

Procedure

1. Make sure the PowerLab is connected

and turned on.
2. Place the pressure pad of the finger
pulse transducer against the distal segment (the
tip) of the middle finger or thumb of either
hand. Use the Velcro strap to attach it firmly -
neither loose nor tight.

If the strap is too loose, the signal will be

weak, intermittent, or noisy. If the strap is too
tight, this will reduce blood flow to the finger,
thus weakening the signal, and may also cause
discomfort. You may need to adjust the strap in
the next stage of the exercise.
 3. Connect the plug from the finger pulse transducer's cable to the socket for Input 1.

Recording Data

1. Click the Start button in the top right-hand corner of the LabTutor panel to begin
recording your finger pulse data.
2. Click the Autoscale button at the top of the LabTutor panel. The data will now be
scaled to occupy most of the height of the channel. You will use the Autoscale
button frequently to optimize the display of your data.
3. After about 20 seconds, click Stop. The data that you have recorded is
automatically saved when you stop recording.

Scrolling

The scroll bar allows you to move back and forward through your file. You can think of
your recording as a large strip of paper scrolling past the LabTutor panel.

The scroll bar is primarily an analysis tool, allowing you to review and locate data of
interest anywhere within a file.

During recording you can also review your data, without stopping recording, by initiating
Scroll Review mode. Scroll Review mode is engaged by either dragging the scroll bar
thumb to the left or clicking anywhere in the incoming data. To return to normal scrolling
mode click the Move to End of Data button.

Generally, while recording, you will be more interested in examining the incoming signal
than reviewing earlier recorded data.

Horizontal Compression Buttons

With the Horizontal Compression buttons, located at the bottom left of a LabTutor panel,
you can compress or expand the Time axis to see more or less of the recorded data.

Click a few times on the Compression buttons to see what this does to the display of your
data. The extent of compression is displayed as a ratio on the Ratio button, which is
located between the Compression buttons.

Click the Ratio button: a pop-up menu appears; from this you can choose the
compression directly.
Vertical Scaling Buttons

The Vertical Scaling buttons are on the left side of each channel's Amplitude axis. These
buttons allow you to compress or expand the visible part of the vertical scale in each
channel independently.

If you move the pointer over the scale part of the Amplitude axis, small arrows appear
beside the pointer. You can either stretch or move the scale by dragging the scale
numbers or the scale between them. The small arrows beside the pointer indicate what
will happen.

Autoscale and Default Scale Buttons

The Autoscale button adjusts the height of the scaled data to display the minimum and
maximum data. Autoscale adjusts the display based on the data visible on screen.

The Default Scale button resets the vertical scale and horizontal compression to their
original settings. This can be useful if, during the course of your experiment, you lose
focus on your data because you have manipulated the vertical scale beyond the signal
limits.

Annotating a Record

Records can be annotated. A Comment panel is provided under the LabTutor panel for
this purpose when required. You can add comments either while you are recording, or
after you have finished.

Procedure

1. Click Start in the LabTutor panel to start recording.

2. Click in the Comment panel text-box, and type in some text.
3. Click the Add button. The text disappears and a vertical dotted line appears in the
LabTutor panel.
4. Repeat steps 2 and 3 to add a second comment.
5. Click Stop.

After you have finished recording, you will see numbered Comment boxes in the
LabTutor panel.
Now try these:

• Click a Comment box: the text you typed appears in the pop-up panel. Edit the
comment by typing new text into the comment panel and clicking the Edit button.
• Add comments after recording has finished. Click in the LabTutor panel at the
place where you wish to insert the comment and then proceed as outlined in steps
2 and 3 above.

Making Measurements

You can make measurements and add the relevant numbers into a Table.

1. Place the cursor over the data at the desired point and click to place the selected
data in the Value panel.
2. To insert a value into a table, drag it from the Value panel into the appropriate cell
of the table.
3. You can also type information, including values, into any cell in the Table.
4. Complete the Table using the data you have already recorded, by clicking a peak
of the pulse recording and transferring the time and amplitude values into the first
row of the table. Repeat for the next three peaks of the pulse.

The Marker

When the Marker is in use, values and times displayed in the Value panel are relative to
the Marker position. When not in use, the Marker resides in a dock at the bottom left of
the LabTutor panel; from here it can be dragged and dropped on to any part of the data.
To return the Marker to its dock, simply click in the dock area.

1. Drag the Marker from its home and drop it somewhere on the trace. The Marker
does not have to be placed exactly on the waveform. When released, the Marker
will drop and attach itself to the waveform.

Values placed in a Value panel will now be prefaced by a Δ (delta) symbol, to

indicate that the measurement is relative to the Marker's position.

2. Complete the Table using the Marker. Place the Marker on a peak of the pulse
recording. Click on the following trough to add the values for time and amplitude
to their respective Value panels. Drag these values into the table and repeat for the
next three peaks of the pulse (see example).
3. Remove the Marker from the waveform by clicking in the Marker dock.
Calculations

LabTutor can be configured to calculate variables based on the raw signal input from
other channels. These can be displayed in real time on channels you are not using for data
acquisition.

Procedure

1. Click Start in the LabTutor panel to start recording.

2. Click in the Comment panel text-box, and type in the volunteer's name.
3. Observe the following traces as they appear on the screen:
o Channel 1 is the finger pulse
o Channel 2 displays the interval between peaks of the pressure waves.
o Channel 3 displays the calculated heart rate in Beats Per Minute.
4. Click Stop

When more than one channel is displayed:

• You can change the channel heights by dragging the channel separators up or
down.

Deleting Data

LabTutor automatically saves your data. Occasionally you may want to discard a segment
of your trace or delete some noisy data.

This action cannot be undone!

Procedure

1. Scroll through the data you just recorded and find a section that appears
excessively noisy. Click the Auto Scale or Default Scale button, as necessary.
2. Click and drag over the portion of the trace you want to remove.

You will notice that LabTutor automatically selects the corresponding data from
all channels displayed on the screen. You cannot delete data from a single
channel; this ensures that the time record always corresponds to the voltage value
recorded.

3. Click the Clear Selection button.

If you select a portion of data from the middle of a recording, LabTutor will insert a
vertical black line into the trace, indicating that the data has been broken into separate
records. You may want to insert a comment at this break to indicate that data was deleted.
 EXERCISE I

HEMOLYSIS AND CRENATION

Learning Objectives:

1. To observe the changes undergone by red blood cells when mixed with the
following solutions: distilled water, 0.9% NaCl, and 3% NaCl solution.
2. To discuss the mechanism of such changes of red cells with the above different
solutions.
3. To differentiate hemolysis from crenation.

Apparatus and Materials:

Three test tubes, test tube rack, distilled water, 0.9% NaCl, and 3% NaCl solution

Procedure:

1. Into each of the three test tubes, place one drop of anticoagulated blood, then add
the following:
a. Test Tube 1 – 5 mL of distilled water
b. Test Tube 2 – 5 mL of 0.9% NaCl
c. Test Tube 3 – 5 mL of 3% NaCl
2. Mix by inversion
3. Avoid shaking the tube violently
4. Set the tube aside and examine after 30 minutes
5. Crenation should be confirmed microscopically.

Observation and Analysis:

Describe the changes that the RBCs underwent with the different solutions.
Explain the mechanism of the changes.
 EXERCISE II

RESPIRATORY AIR FLOW AND VOLUME

Introduction

In this laboratory, you will be introduced to spirometry as a technique for recording

respiratory variables and you will analyze a recording to derive respiratory parameters.
You will examine lung volumes and capacities, as well as the basic tests of pulmonary
function and simulate an airway restriction.

Learning Objectives

By the end of today's laboratory you will be able to:

• Explain the principles of spirometry and how integration of the flow signal gives
a volume.
• Relate your recorded lung volumes and capacities, to those of a typical person of
the same gender, height and age.
• Perform pulmonary function tests, describe the common measurements made
from them (PIF, PEF, FVC and FEV1) and relate these measurements to those of a
typical person of the same gender, height and age.
• Describe the effect of airway restrictions on PIF, PEF, FVC and FEV1.

Spirometer, registering the movements of the chest, the quantity of expired air and the
time during which the phenomena occur.

Respiration apparatus according to Zuntz.

Equipment Setup

Procedure

1. Connect the Spirometer Pod to Input 1 on the PowerLab.

2. Since the Spirometer Pod is sensitive to temperature and tends to drift during
warm-up, turn on the PowerLab for at least 5 minutes before use. To prevent
temperature drift due to heating of the Pod, place it on a shelf or beside the
PowerLab, away from the PowerLab power supply.
3. Connect the two plastic tubes from the respiratory flow head to the short pipes on
the back of the Spirometer Pod.
4. Attach Clean Bore tubing, a filter and mouthpiece to the flow head.
5. Make sure you have access to the following equipment for different parts of the
experiment.
o Tape measure for measuring volunteer height.
o Duct tape and a pen, or sharpened pencil, for the simulated airway
restriction exercise.
o Extra mouthpieces and disposable air filters for each volunteer.

If you are suffering from a respiratory infection, do

not volunteer for this experiment.

Zeroing the Spirometry Pod

The Spirometry Pod is susceptible to thermal drift of

the baseline signal. In order to give appropriate
volume measurements it is important to always reset
the baseline by clicking the Zero Pod button prior to
making any new recording.

Procedure

1. Leave the flow head apparatus undisturbed on the bench and click the Zero Pod
button. This will reset the offset of the Flow channel to zero.
2. Click Start. The volunteer can now put the mouthpiece in his or her mouth, and
hold the flow head carefully with two hands.

In order to calculate volume from the flow recording correctly it is crucial that
recording be started prior to breathing through the flow head.

3. Put the nose clip on the volunteers nose. This ensures that all air breathed passes
through the mouthpiece, filter and flow head.
4. Observe the trace. The signal should show a downward deflection on expiration.
If the signal deflects upward, stop recording and either reverse the orientation of
the flow head, or swap the tubular connections to the Spirometer Pod.
 5. When the volunteer has become accustomed to the apparatus and is breathing
normally through it, stop recording and proceed to the next page.

Volume Correction

Your instructor may not require you to perform this volume correction. Please check
before continuing. If this is the case, proceed to the next page.

Expired air is greater in volume than inspired air under most atmospheric conditions. This
increase, due to warming and humidification, is typically 5-10%. For this reason it is
common to apply a volume correction factor to the Volume channel.

Procedure

1. Re-zero the Spirometry Pod using the Zero Pod button. Remember that the flow
head must be left undisturbed on the bench during the zeroing process.
2. Click Start. Once recording has started ask the volunteer to pick up the flowhead
and start breathing through it.
3. Have the volunteer perform a full expiration through the flowhead and then
continue normal tidal breathing for one minute.
4. While recording continues add the comment "Volume correction procedure" to
the data.
5. At the end of one minute, have the volunteer perform another full expiration.
6. Click Stop. The volunteer can now stop breathing through the flow head and
remove the nose clip.

Analysis

1. Select the entire recording of tidal breathing data, including the two forced
expirations by double-clicking in the Time axis beneath the trace (this selects a
record).
2. The default value for the volume correction factor is 1.08. Once you have selected
the appropriate data, LabTutor will suggest a new volume correction factor. If you
wish to accept this value click the Apply button.

Exercise 1

In this exercise, you will examine the respiratory cycle and measure changes in volume.

It is important when recording normal respiration that the volunteer is not consciously
controlling breathing. The volunteer should turn away from the computer screen and may
have to stare out a window or read a book to distract themselves.
Procedure

1. Re-zero the Spirometer Pod using the Zero Pod button. Remember that the flow
head must be left undisturbed on the bench during the zeroing process.
2. Click Start. Once recording has started ask the volunteer to replace the nose clip
and breathe normally through the flow head. Record normal tidal breathing for 1
to 2 minutes.
3. During recording add the comment "Normal tidal breathing" to the data.
4. After the tidal breathing period and at the end of a normal tidal expiration, ask the
volunteer to inhale as deeply as possible and then exhale as deeply as possible.
Afterwards, allow the volunteer to return to normal tidal breathing then stop
recording.
5. Add the comment "Lung volume procedure" to this deep breath.

Analysis

Follow the steps below to complete the table. (Table 2-1)

1. Examine the normal tidal breathing data. Calculate how many breaths there are in
a one-minute period (bpm). Type this into the appropriate cell in the table.
2. Determine the volume of a single tidal inspiration by dragging the Marker from
its box to the Volume channel at the start of a normal tidal inspiration. Move the
Waveform Cursor to the next peak on the Volume channel (this should be 0.5 to
1.5 s to the right of the Marker).
3. Click to place the selected data in the Value panel and drag the value from the
Value panel into the Tidal Volume (VT) cell of the table. Expired minute volume
will be calculated by LabTutor for you.
4. Repeat steps 2-3 to determine the Inspiratory Reserve Volume (IRV) and
Expiratory Reserve Volume (ERV). Note, the Marker should remain at the start of
a normal tidal inspiration (trough) for the ERV procedure, it should be moved to
the end of a normal tidal inspiration (peak) for for the IRV procedure.
5. Click on the link and use the calculator to determine predicted values for Residual
Volume (RV).

Calculate the lung capacities.

Study Questions

1. Comment on the differences between the experimental and predicted values for
VC, FRC and TLC in the table above. What could cause these differences, if any?

2. In quiet breathing, muscular effort is used mainly in inspiration, and expiration is

largely passive, due to elastic recoil of the lung. Can you relate this fact to the
pattern of expiratory and inspiratory flow? Hint: the normal pattern of breathing is
efficient in that it requires muscular effort for only a short time.

3. Explain why RV cannot be determined by ordinary spirometry?

Exercise 2

In this exercise, you will measure parameters of forced expiration that are used in
evaluating pulmonary function

You should use the same volunteer as for exercise 1.

Procedure

1. Re-zero the Spirometer Pod using the Zero Pod button. Remember that the flow
head must be left undisturbed on the bench during the zeroing process
2. Click Start. Once recording has started ask the volunteer to replace the nose clip
and breathe normally through the flow head.
3. Prepare a comment "FVC procedure".
4. Have the volunteer breathe normally for 10 to 20 seconds.
5. Ask the volunteer to inhale and then exhale as forcefully, as fully and for as long
as possible, until no more air can be expired.
6. In the comment box, click Add.
7. Allow the volunteers breathing to return to normal, then click Stop.

8. Repeat this procedure twice more, so that you have three separate Forced Vital
Capacity recordings.

Analysis

Follow the steps below to complete the table. (Table 2-2)

1. Using the Waveform Cursor and the Marker tool as necessary, examine each of
the three Forced Vital Capacity recordings (FVC).
2. On the Flow channel determine which of the three recordings shows a maximum
Peak Inspiratory Flow (PIF).
3. Click to place this data in the Value panel. Drag this value into the appropriate
cell in the table.
4. Repeat this step to determine the maximum Peak Expiratory Flow (PEF), and
enter this into the table also.
5. On the Volume channel determine which of the three recordings shows a maximal
FVC.
6. Place the Marker on the peak inhalation in the Volume channel and move the
Waveform Cursor to the maximal expiration also on the Volume channel. Click to
place the selected data in the Value panel, and drag the value from the Value
panel into the FVC cell of the table.
7. Using the same recording as gave a maximal FVC measure the Forced Expired
Volume in 1 second (FEV1). Place the Marker on the peak inhalation in the
Volume channel, move the pointer to a time 1.0 s from the peak. Click to place
the selected data in the Value panel, and drag the value from the Value panel into
the FVC cell of the table.
The ratio of FEV1 to FVC expressed as a percentage, will be calculated by LabTutor for
you.

Study Questions

1. Comment on the differences between the experimental and predicted values for
FVC, FEV1 and the FEV1/FVC ratio in the table above. What could cause these
differences, if any?

2. In you own words describe the physiological significance of the FEV1/FVC ratio?

3. Were your results for forced breathing consistent across all three trials? If not,
why not?
Exercise 3

The effects of bronchial restrictions such as asthma can be demonstrated by making the
following modification to the equipment.

Setup

1. Remove the filter attachment from the clean-bore tubing.

2. Cover the end of the filter with duct tape.
3. Use a pen or a sharpened pencil to make a hole in the duct tape over the filter
about a half centimeter in diameter.
4. Reattach the filter to the clean-bore tubing.

Procedure

Repeat the procedures in Exercise 2, as described below.

1. Re-zero the Spirometer Pod using the Zero Pod button. Remember that the flow
head must be left undisturbed on the bench during the zeroing process
2. Click Start. Once recording has started ask the volunteer to replace the nose clip
and breathe normally through the flow head.
3. Prepare a comment "FVC restricted".
4. Have the volunteer breathe normally for 10 to 20 seconds.
5. Ask the volunteer to inhale and then exhale as forcefully, as fully and for as long
as possible, until no more air can be expired.
6. In the comment box, click Add.
7. Allow the volunteers breathing to return to normal, then click Stop.
8. Repeat this procedure twice more, so that you have three separate Forced Vital
Capacity recordings.

Analysis

Repeat the analysis in Exercise 2, as described below. (Table 2-3)

1. Using the Waveform Cursor and the Marker tool as necessary, examine each of
the three Forced Vital Capacity recordings (FVC).
2. On the Flow channel determine which of the three recordings shows a maximum
Peak Inspiratory Flow (PIF).
3. Click to place this data in the Value panel. Drag this value into the appropriate
cell in the table.
4. Repeat this step to determine the maximum Peak Expiratory Flow (PEF), and
enter this into the table also.
5. On the Volume channel determine which of the three recordings shows a maximal
FVC.
 6. Place the Marker on the peak inhalation in the Volume channel and move the
Waveform Cursor to the maximal expiration also on the Volume channel. Click to
place the selected data in the Value panel, and drag the value from the Value
panel into the FVC cell of the table.
7. Using the same recording as gave a maximal FVC measure the Forced Expired
Volume in 1 second (FEV1). Place the Marker on the peak inhalation in the
Volume channel, move the pointer to a time 1.0 s from the peak. Click to place
the selected data in the Value panel, and drag the value from the Value panel into
the FVC cell of the table.

The ratio of FEV1 to FVC expressed as a percentage, will be calculated by LabTutor for
you.

Study Questions

1. Based on your data, what values have been affected by simulated airway
restriction and why?

2. In your own words explain the physiological events that occurred during this
simulated asthma attack.
Hint: Think about what it felt like and how that would affect your general state of
well being and activity level.
Exercise 4

In this exercise, you will compare the parameters of forced expiration measured in
different volunteers.

Procedure

Repeat the procedures in Exercise 2, as described below, for up to three more volunteers.

Remember to replace the disposable filter and mouthpiece for each new volunteer.

1. Re-zero the Spirometer Pod using the Zero Pod button. Remember that the flow
head must be left undisturbed on the bench during the zeroing process
2. Click Start. Once recording has started ask the volunteer to replace the nose clip
and breathe normally through the flow head.
3. Prepare a comment "FVC volunteer 2".
4. Have the volunteer breathe normally for 10 to 20 seconds.
5. Ask the volunteer to inhale and then exhale as forcefully, as fully and for as long
as possible, until no more air can be expired.
6. In the comment box, click Add.
7. Allow the volunteers breathing to return to normal, then click Stop.
8. Repeat this procedure up to twice more, so that you have three separate Forced
Vital Capacity recordings.
9. Repeat for up to another two volunteer, .

Analysis

Repeat the analysis in Exercise 2 & 3 for each volunteer, as described below.(Table 2-4)

1. Using the Waveform Cursor and the Marker tool as necessary, examine each of
the three Forced Vital Capacity recordings (FVC).
2. On the Flow channel determine which of the three recordings shows a maximum
Peak Inspiratory Flow (PIF).
3. Click to place this data in the Value panel. Drag this value into the appropriate
cell in the table.
4. Repeat this step to determine the maximum Peak Expiratory Flow (PEF), and
enter this into the table also.
5. On the Volume channel determine which of the three recordings shows a maximal
FVC.
6. Place the Marker on the peak inhalation in the Volume channel and move the
Waveform Cursor to the maximal expiration also on the Volume channel. Click to
place the selected data in the Value panel, and drag the value from the Value
panel into the FVC cell of the table.
 7. Using the same recording as gave a maximal FVC measure the Forced Expired
Volume in 1 second (FEV1). Place the Marker on the peak inhalation in the
Volume channel, move the pointer to a time 1.0 s from the peak. Click to place
the selected data in the Value panel, and drag the value from the Value panel into
the FVC cell of the table.

The ratio of FEV1 to FVC expressed as a percentage, will be calculated by LabTutor for
you.

Study Questions

1. Comment on the range of results shown in the table for Exercise 4.

2. What factors do you think could contribute to differences in pulmonary

parameters between the volunteers?
 EXERCISE III

MECHANICS OF VENTILATION

Introduction

In this laboratory, you will examine mechanical properties of the lung and chest wall by
measuring pressures generated passively and by contraction of expiratory and inspiratory
muscles. You will also be introduced to spirometry as a technique for determining lung
volume.

Hermann Rahn (1912-1990). Wallace O. Fenn (1893-1971).

Rahn and Fenn contributed to the classic paper about lung mechanics in 1946. The
figure shows the original pressure volume graph from the paper Rahn H, Otis AB,
Chadwick, LE & Fenn, WO (1946). The pressure - volume diagram of the thorax and
lung. Am. J. Physiol.146: 161-178.
Learning Objectives

By the end of today's laboratory you will be able to:

• Describe the relationship between lung volume and pressure during breathing.
• Describe the elastic properties of the chest wall, lungs and the contractile muscles
that inflate the lungs.
• Define terminology associated with the mechanics of ventilation.
• Estimate your participant's Vital Capacity (VC), Lung Volumes and Compliance.

PowerLab Setup

Spirometer Pod

In order to measure the pressures generated at different lung volumes you will use
techniques of spirometry.

• Connect the Spirometer Pod to Input 1 on the PowerLab.

Since the Spirometer Pod is sensitive to temperature and tends to drift during warm-
up, turn on the PowerLab for at least 5 minutes prior to use. To prevent temperature drift
due to heating of the Pod, place it on a shelf or beside the PowerLab, away from the
PowerLab power supply.

Pressure transducer

Pressure will be measured using the transducer from a

blood pressure cuff.

• Connect the pressure transducer to Input 2 on the

PowerLab.

Equipment Setup

To measure and compare the pressures generated at different lung volumes you need to
setup the equipment illustrated using the steps below.

Volume Measurement

1. Connect the two plastic tubes from the respiratory flow head to the short pipes on
the back of the Spirometer Pod.
2. Attach a mouthpiece, filter and clean-bore tubing to the three-way tap.
3. Connect the flow head to the three-way tap. Make sure a plug is inserted into the
three-way tap outlet to prevent air flow.
Pressure Measurement

Pressure will be measured using the transducer from a blood pressure cuff.

1. Connect the sampling tube from the pressure transducer to the sampling port on the
three-way tap.
2. Ensure an adaptor is in place, to block air exiting from the remaining outlet on the
three-way tap.

2. Click Start, breathe out through the mouthpiece

then click Stop.

You should see a positive response from the

upper Flow channel and an increase in Volume
from the lower channel. If not, it's likely the connectors from the flow head to the
Spirometer Pod are reversed. Change these over at the Spirometer Pod and repeat
the above procedure.

Vital Capacity

In order to familiarize yourself with the operation of the flow head you will determine
your volunteer's vital capacity (VC). First, you will need to check your equipment setup.

If you are suffering from a respiratory infection, do not volunteer for this experiment.

Equipment Check

1. Zero the Spirometer Pod using the Zero Pod button below. The flow head must be
left undisturbed on the bench during the zeroing process.

2. Click Start, breathe out through the mouthpiece then click Stop.

You should see a positive response from the upper Flow channel and an increase in
Volume from the lower channel. If not, it's likely the connectors from the flow head to
the Spirometer Pod are reversed. Change these over at the Spirometer Pod and repeat the
above procedure.
Recording

1. Ensure the three-way tap is open to the flow head.

2. Zero the Spirometer Pod as above.
3. Place a nose clip on the volunteer and instruct him or her to inhale maximally from
room air (100% of VC).
4. Click Start.
5. Ask the volunteer to exhale as much as possible (to residual volume (RV)) through
the mouthpiece to the flow head.
6. Click Stop.
7. Add a Comment 'Vital Capacity' to the data.
8. Repeat this process until a consistent readout is given (average values ~ 3200 -
4800 mL).

Analysis

1. In the LabTutor panel, double click on the time axis to select a record block. The
volunteer's VC value will appear in the Value panel.
2. Drag this value into the cell in the table. (TABLE 3-1)

Expiratory Pressure

In this procedure you will measure the expiratory pressures of the lung and chest wall at
different volumes. Ensure the volunteer is wearing a nose clip at all times.

Procedure

Pressure at 100% VC

1. Zero the Spirometer Pod using the Zero Pod button. The flow head must be left
undisturbed on the bench during the zeroing process.
2. Ensure the three-way tap is open to the pressure transducer.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC), insert the
mouthpiece and then, exhale maximally

Note that no air will flow, as there is a closed loop between the lungs and chest wall.
You should see a positive pressure.

6. Click Stop.
7. Add the Comment 'Expiratory pressure, VC' to the data.
Pressure at lower volumes

In order to measure expiratory pressure at different lung volumes, you will repeat the
procedure above, but depleting lung volume in 500 mL steps.

Note: The volunteer needs to breath out slowly during the volume depletion, so the
expired volume can be measured accurately. You may need to practice the timing of this
exercise a couple of times to get accurate volume depletions.

1. Zero the Spirometer Pod, as above.

2. Ensure the three-way tap is open to the flow head.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC).
6. Ask the volunteer to breathe out slowly to the flow head.
7. Monitor the volume of expired air in the Readout Panel. Once ~500 mL has been
exhaled, switch the three-way tap to the pressure transducer, preventing air flow.
8. Instruct the volunteer to exhale maximally again, as above.
9. Click Stop.
10. Add the Comment 'Expiratory pressure, -500 mL' to the data.

Repeat this volume depletion increasing the amount breathed out by 500 mL each time
(500 mL, 1000 mL, 1500 mL, etc...), until residual volume is reached.

Analysis

Use the Horizontal Compression buttons and the scroll bar, to review the data obtained
during the test. (TABLE 3-2)

1. Display the first record of expiratory pressure in the LabTutor Panel. This will be
your recording of the volunteer's Vital Capacity (VC).
2. Select the entire record of the Expiratory pressure VC measurement. Values for
Volume and Pressure will appear in the Value panels. Note: you have already
measured your volunteer's VC (volume).
3. Drag the pressure value into the appropriate cell in the table.
4. Now, select the second recording (500 mL depleted).
5. Drag the values for Volume and Pressure from the Value panels into the appropriate
cells in the table.
6. Repeat steps 4 and 5 for the remaining Expiratory recordings.

Note that the first recording should have been at 100% vital capacity, so no volume
change entry is necessary.
Inspiratory Pressure

In this procedure you will measure the inspiratory pressures of the lung and chest wall at
different volumes. Ensure the volunteer is wearing a nose clip at all times.

Procedure

Pressure at 100% VC

1. Zero the Spirometer Pod as above.

2. Ensure the three-way tap is open to the pressure transducer.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC), insert the
mouthpiece and then, inhale maximally.

Note that no air will flow, as there is a closed loop between the lungs and chest
wall. You should see a negative pressure.

6. Click Stop.
7. Add the Comment 'Inspiratory pressure, VC' to the data.

Pressure at lower volumes

In order to measure inspiratory pressure at different lung volumes, you will repeat the
procedure above, but depleting lung volume in 500 mL steps.

Note: the volunteer needs to breath out slowly during the volume depletion, to ensure
it is accurately measured. You may need to practice the timing of this exercise a couple
of times to get accurate volume depletions.

1. Zero the Spirometer Pod, as above.

2. Ensure the three-way tap is open to the flow head.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC).
6. Ask the volunteer to breathe out slowly to the flow head.
7. Monitor volume on the Readout Panel. Once ~500 mL has been exhaled, switch the
three-way tap to the pressure transducer.
8. Instruct the volunteer to inhale maximally again, as above.
9. Click Stop.
10. Add the Comment 'Inspiratory pressure, -500 mL' to the data.

Repeat this volume depletion increasing the amount breathed out by 500 mL each time
(500 mL, 1000 mL, 1500 mL, etc...), until residual volume is reached.
Analysis

Use the Horizontal Compression buttons and the scroll bar, to review the data obtained
during the test. (TABLE 3-3)

1. Display the first record of expiratory pressure in the LabTutor Panel. This will be
your recording of the volunteer's Vital Capacity (VC).
2. Select the entire record of the Inspiratory pressure VC measurement. Values for
Volume and Pressure will appear in the Value panels. Note: you have already
measured your volunteer's VC (volume).
3. Drag the pressure value into the appropriate cell in the table.
4. Now, select the second recording (500 mL depleted).
5. Drag the values for Volume and Pressure from the Value panels into the appropriate
cell in the table.
6. Repeat steps 4 and 5 for the remaining Inspiratory recordings.

Note that the first recording should have been at 100% vital capacity, so no volume
change entry is necessary.

Passive Pressure

In this procedure you will measure the passive recoil pressures of the lung and chest wall
at different volumes.

It can be difficult to get correct passive pressure results from an untrained volunteer.
Attempt the procedure below. Representative data from a trained individual has been
included in your tables and is plotted automatically to allow you to compare your
inspiratory and expiratory pressures. Ensure the volunteer is wearing a nose clip at all
times.

Procedure

Pressure at 100% VC

1. Zero the Spirometer Pod using the Zero Pod button below. The flow head must be
left undisturbed on the bench during the zeroing process.
2. Ensure the three-way tap is open to the pressure transducer.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC), insert the
mouthpiece and then, relax their chest with their glottis open.

Note that no air will flow, as there is a closed loop between the lungs and chest
wall. You should see a positive pressure at volumes above functional residual
capacity (FRC) and negative at volumes below FRC.
 6. Click Stop.
7. Add the Comment 'Passive pressure, VC' to the data.
Pressure at lower volumes

In order to measure passive pressure at different lung volumes you will repeat the
procedure above except depleting lung volume in 500 mL steps.

Note: the volunteer needs to breath out slowly during the volume depletion, to ensure
it is accurately measured. You may need to practice the timing of this exercise a couple
of times to get accurate volume depletions.

1. Zero the Spirometer Pod, as above.

2. Ensure the three-way tap is open to the flow head.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC).
6. Ask the volunteer to breathe out slowly to the flow head.
7. Monitor volume on the Readout Panel. Once they have exhaled ~500 mL switch the
three-way tap to the pressure transducer.
8. Instruct the volunteer to relax the chest, as above.
9. Click Stop.
10. Add the Comment 'Passive pressure, -500 mL' to the data.

Repeat this volume depletion but increase the amount breathed out by 500 mL each time
(500 mL, 1000 mL, 1500 mL, etc...), until residual volume is reached.

Analysis

If you were able to obtain results for the passive pressure exercise follow the instructions,
otherwise move on to the next page.

Use the Horizontal Compression buttons and the scroll bar, to review the data obtained
during the test. (TABLE 3-4)

1. Display the first record of expiratory pressure in the LabTutor Panel. This will be
your recording of the volunteer's Vital Capacity (VC).
2. Select the entire record of the Passive pressure VC measurement. Values for
Volume and Pressure will appear in the Value panels. Note: you have already
measured your volunteer's VC (volume).
3. Drag the pressure value into the appropriate cell in the table.
4. Now, select the second recording (500 mL depleted).
5. Drag the values for Volume and Pressure from the Value panels into the appropriate
cell in the table.
6. Repeat steps 4 and 5 for the remaining Inspiratory recordings.
Passive Lung Pressure

Procedure

Pressure at 100% VC

1. Zero the Spirometer Pod, as above.

2. Ensure the three-way tap is open to the pressure transducer.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC), insert the
mouthpiece and then, open the glottis, but this time, instead of relaxing the chest,
hold the respiratory muscles in a sustained contraction so that lung volume remains
constant.

Note that again, no air will flow. Since the respiratory muscles are not relaxed, they
are not producing any recoil forces; only the lungs are recoiling.

6. Click Stop.
7. Add the Comment 'Passive lung pressure, VC' to the data.

Pressure at lower volumes

In order to measure passive pressure at different lung volumes you will repeat the
procedure above except depleting lung volume in 500 mL steps.

Note: the volunteer needs to breath out slowly during the volume depletion, to ensure
it is accurately measured. You may need to practice the timing of this exercise a couple
of times to get accurate volume depletions.

1. Zero the Spirometer Pod, as above.

2. Ensure the three-way tap is open to the flow head.
3. Place a nose clip on the volunteer.
4. Click Start.
5. Instruct the volunteer to inhale maximally from room air (100% of VC).
6. Ask the volunteer to breathe out slowly to the flow head.
7. Monitor volume on the Readout Panel. Once they have exhaled ~500 mL switch the
three-way tap to the pressure transducer.
8. Instruct the volunteer to open the glottis but not to relax the respiratory muscles,
as above.
9. Click Stop.
10. Add the Comment 'Passive pressure, -500 mL' to the data.

Repeat this volume depletion but increase the amount breathed out by 500 mL each time
(500 mL, 1000 mL, 1500 mL, etc...), until residual volume is reached.
Analysis

If you were able to obtain results for the passive pressure exercise follow the instructions,
otherwise move on to the next page.

Use the Horizontal Compression buttons and the scroll bar, to review the data obtained
during the test. (TABLE 3-5)

1. Display the first record of expiratory pressure in the LabTutor Panel. This will be
your recording of the volunteer's Vital Capacity (VC).
2. Select the entire record of the Passive pressure VC measurement. Values for
Volume and Pressure will appear in the Value panels. Note: you have already
measured your volunteers VC (volume).
3. Drag the pressure value into the appropriate cell in the table.
4. Now, select the second recording (500 mL depleted).
5. Drag the values for Volume and Pressure from the Value panels into the appropriate
cell in the table.
6. Repeat steps 4 and 5 for the remaining Inspiratory recordings.

Note that the first recording should have been at 100% vital capacity, so no volume
change entry is necessary.
 EXERCISE IV

BREATHING

Introduction

In this laboratory, you will record breathing movements with a respiratory belt transducer
fastened around the abdomen. You will investigate various aspects of breathing,
including the ability to hold the breath, hyperventilation, rebreathing, and the relation
between breathing and heart rate.

Learning Objectives

By the end of today's laboratory you will be able to describe and explain the:

• Effects on breathing of voluntary hyperventilation

• Effects on the respiratory pattern of rebreathing expired gas
• Relationship between breathing and heart rate

This device was developed by the French scientist

Étienne- Jules Marey (1830-1904) to indicate An example of an early
movements of the chest due to breathing. It is spirometer: Hutchinsons
mounted on a spring plate which returns to its spirometer.
original position when the breath is expired.
Procedure

1. Fasten the respiratory belt around the abdomen of a volunteer, as shown. The
transducer should be:
o At the front of the body, level with the navel.
o Tightened sufficiently that it remains under tension even when the subject
fully exhales.

The respiratory belt transducer can be used over clothing, and it doesn't matter whether
the volunteer is sitting or standing, so long as they are comfortable (this is quite a long
exercise). Because breathing patterns differ, you may need to reposition the transducer
over the chest rather than the abdomen to get the best signal.

2. Connect the plug on the respiratory belt transducer cable to Input 1 on the front of
the PowerLab.

It is important when recording normal respiration that the volunteer is facing away
from the computer screen and is not consciously controlling breathing. The volunteer
may have to stare out a window or read a book to avoid conscious control of respiration.
Exercise 1

In this exercise, you will record normal and rapid breathing, and examine the effects of
holding your breath after inhaling and after exhaling.

Procedure

1. Click Start.
2. Ask the volunteer to breathe rapidly for a few seconds, and then to breathe slowly.
Examine the Breath Rate channel, there should be obvious changes in rate.
3. Enter a comment: 'Baseline 1' in the slow breathing region of the data.
4. Click Add.
5. Record 2-3 minutes of normal, quiet breathing and observe the trace.
6. Enter a comment: 'inhale, hold'.
7. Click Add, and immediately ask the volunteer to take a deep breath and hold it in
for as long as possible.
8. Enter a comment: 'breathe'.
9. When the volunteer begins to breathe again, click Add .
10. Wait until a normal (baseline) breathing pattern resumes; then let the volunteer
rest and breathe normally for another 2-3 minutes.
11. Enter a comment: 'exhale, hold'.
12. Click Add, and immediately ask the volunteer to breathe out fully and hold the
breath for as long as possible.
13. Enter a comment: 'breathe'.
14. When the volunteer begins breathing click Add.
15. Continue recording until a normal (baseline) pattern resumes.
16. Click Stop.

The volunteer can now relax and breathe normally.

Analysis (TABLE 4-1)

1. In the Breathing channel, place the Marker on the large peak following the
comment 'inhale, hold'.
2. Move the Waveform Cursor to the start of the first breath afterwards, which
should be preceded by the comment 'breathe'.
3. Click to place the selected data in the Value panel and drag it from the Value
panel into the appropriate cell of the table.
4. Drag the Marker to the large (negative) peak right after the comment 'exhale,
hold'.
5. Move the Waveform Cursor to the start of the first breath afterwards, which
should also be preceded by the comment 'breathe'.
6. Click to place the selected data in the Value panel and drag it from the Value
panel into the appropriate cell of the table.
Exercise 2

In this exercise, you will record the effect of voluntary hyperventilation on breath-
holding and the recovery of normal breathing rhythm.

Procedure

1. Click Start.
2. Enter a comment 'baseline', click Add and ask the volunteer to maintain normal
respiration for 2-3 minutes.
3. Enter a comment: 'inhale, hold'.
4. Click Add, and immediately ask the volunteer to take a deep breath and hold it in
for as long as possible.
5. Enter a comment: 'breathe'.
6. When the volunteer begins to breathe again, click Add .
7. Record the subject's normal respiration for 2-3 minutes. During this time, enter a
comment: 'hyperventilate'.
8. Click Add and immediately ask the volunteer to hyperventilate by breathing as
quickly and as deeply as possible for 30 seconds.
9. Enter a comment: 'breathe'.
10. After the 30 seconds of hyperventilation click Add, then immediately tell the
volunteer to begin breathing normally again.
11. Wait until a normal breathing pattern resumes; then let the volunteer rest and
breathe normally for another 2-3 minutes.
12. Enter a comment: 'hyperventilate'.
13. Click Add, then immediately ask the volunteer to hyperventilate again by
breathing as quickly and as deeply as possible for 30 seconds.
14. Enter a comment: 'inhale, hold'.
15. After the 30 seconds of hyperventilation click Add, and Immediately ask the
volunteer to take a deep breath and hold it in for as long as possible.
16. Enter a comment: 'breathe'.
17. When the volunteer begins breathing click Add.
18. Click Stop.

The volunteer can now relax and breathe normally.

Analysis (TABLE 4-2)

1. In the Breath Rate channel, select an area representing your normal breathing rate
prior to the 'inhale, hold' comment. This will give you the average breathing rate
for that period of time.
2. Drag the the Breath Rate data from the Value Panel into the appropriate cell of the
table.
 3. In the Breathing channel, select the period of time for which the subject held their
breath (from the 'inhale, hold' comment to the 'breathe' comment), and drag this
Breathing Selection Duration data from the Value Panel to the appropriate cell in
the table.
4. Repeat steps 1-3 for the hyperventilation period and the subsequent breath-hold.

Exercise 3

In this exercise, you will observe the effect of rebreathing exhaled gases. You will need
to obtain a medium-sized paper bag. When re-breathing, the volunteer should place this
so that it covers the nose and mouth and forms a tight seal.

Procedure

1. Click Start. Enter a comment: 'baseline', and click Add.

2. Record the baseline for 2-3 minutes.
3. Enter a comment: 'rebreathing'.
4. Click Add and immediately ask the volunteer to place the paper bag over the nose
and mouth, and rebreathe the air in the bag.
5. Enter a comment: 'breathe'.
6. After 60 seconds of rebreathing, click Add; then immediately ask the volunteer to
remove the paper bag from the nose and mouth.
7. Continue recording for 60 seconds.
8. Click Stop.

Analysis (TABLE 4-3)

Rebreathing from a closed bag results in arterial hypercapnia (raised partial pressure of
carbon dioxide), which stimulates respiration. How was this evident in this exercise?
(That is, did the depth or rate or both increase during rebreathing compared to normal
breathing?)

Procedure

1. Leave the respiratory belt fastened around the abdomen of the volunteer.
2. Connect the finger pulse transducer to Input 2 on the PowerLab.
3. Place the pressure pad of the finger pulse transducer against the tip of the middle
finger of either hand of the volunteer. Use the Velcro strap to attach it firmly -
neither loose nor tight.
4. Ensure that the person sits quietly with his or her hands resting in their lap, or on a
bench, to minimize transducer movements.
Exercise 4

In this exercise, you will record and examine the effect of breath-holding on heart rate.

Procedure

1. Click Start.
2. Record a baseline heart rate and breathing pattern for two minutes. (Variation in
the heart rate is most evident with slow, deep breathing.)
3. After recording the baseline signals, enter a comment: 'inhale, hold'.
4. Click Add, and immediately ask the volunteer to take a deep breath and hold it in
for as long as possible.
5. While the volunteer is not breathing, enter a comment: 'breathe'.
6. When the volunteer begins breathing, click Add.
7. Click Stop.

The volunteer can now relax and breathe normally.

Analysis (TABLE 4-4)

Heart rate variations within the breathing cycle should be seen best at a timescale
compression of 20:1
 EXERCISE V

BLOOD PRESSURE

Introduction

In this laboratory, you will become familiar with auscultation (listening to the sounds of
the body) and the measurement of blood pressure. The exercises involve measuring your
blood pressure using a stethoscope, blood pressure cuff and sphygmomanometer. You
will also assess changes in peripheral circulation and the effects of cuff location.

The modern era of blood pressure measurement started with the introduction of the
mercury sphygmomanometer by Scipione Riva-Rocci (1863-1937) in 1896.

Riva-Rocci style sphygmomanometer based on the original 1896 design.

Learning Objectives

By the end of today's laboratory you will be able to:

• Use a sphygmomanometer and stethoscope to measure human arterial blood

pressure
• Determine systolic blood pressure using a sphygmomanometer and detection of a
peripheral pulse
• Demonstrate how measurement position affects the magnitude of the arterial
blood pressure

Procedure

To perform this experiment correctly, you must be familiar with the use of the
stethoscope and sphygmomanometer.

The sphygmomanometer conveniently combines a cuff and bulb with a pressure

transducer.
 1. Plug the pressure transducer into Input 1 on the PowerLab.
2. Plug the Cardio Microphone into Input 2.
3. Wrap the sphygmomanometer cuff around the upper arm just above the elbow as
shown.
4. Click Start.
5. Practice inflating the cuff to approximately 180 mmHg and slowly reducing the
pressure (1-2 mmHg per second) until you are confident that you can use the
sphygmomanometer correctly.
6. Click Stop.

Exercise 1

In this exercise you will measure blood pressure in the traditional way, using a
stethoscope to listen for Korotkoff sounds.

Procedure

1. Inflate the cuff until the pressure reaches approximately 180 mmHg.
2. Slowly reduce the pressure in the cuff (approximately 1 to 2 mmHg per second)
while listening through the stethoscope for Korotkoff sounds.
3. The systolic pressure is the pressure at which sharp, tapping sounds are first
heard.
4. Continue slowly reducing cuff pressure (at 1 to 2 mmHg per second). The
diastolic pressure is defined as the pressure at which the sounds disappear.
5. Completely deflate the cuff once diastolic pressure is determined. Do not leave
the cuff partially inflated or leave it inflated for a long time.
6. For each subject, record four measurements of the blood pressure. Allow one to
two minutes between measurements for recovery.
7. Repeat the procedure using other students until you feel confident in measuring
blood pressure. (TABLE 5-1)

Exercise 2

In this exercise you will use the Cardio Microphone to record arterial sound while
recording blood pressure.

Procedure:

1. Leave the blood pressure cuff in place around the upper portion of the student's
arm (either arm), between the elbow and the shoulder.
2. Place the Cardio Microphone over the brachial artery and under the blood
pressure cuff so that it is held in position by the cuff.
3. Click Start.
4. Inflate the cuff until the pressure reaches approximately 180 mmHg.
5. Slowly reduce the pressure in the cuff (approximately 1 to 2 mmHg per second).
Deflate the cuff completely once the pressure has gone below 50 mmHg.
 6. Click Stop.
7. Repeat the procedure using other students. Remember to add a comment with the
subject's name for later identification. Allow one to two minutes between
procedures for recovery.

Analysis (TABLE 5-2)

1. Examine your recording. The Cardio Microphone channel displays the Korotkoff
sounds as spikes. These spikes can be used to determine systolic and diastolic
pressure.
2. Place the Waveform Cursor on the first spike following the reduction in cuff
pressure. This represents the systolic pressure.
3. Click on this point to enter the pressure in the value panel and add the comment
"systolic pressure" to the data.
4. Drag the number from the value panel into the appropriate column of the table.
5. Place the Waveform Cursor on the last spike in the series. This represents the
diastolic pressure.
6. Click on this point to enter the pressure in the value panel and add the comment
"diastolic pressure" to the data.

In some subjects it is not possible to determine diastolic pressure using this

technique.

7. Repeat these procedures for all subjects in your group.

Exercise 3

You will observe the changes in finger pulse while measuring blood pressure, and see if
pulse measurement could replace the use of the stethoscope.

Procedure

1. Remove the Cardio Microphone plug from Input 2 on the PowerLab.

2. Connect the finger pulse transducer to Input 2.
3. Place the pressure pad of the finger pulse transducer against the distal segment
(the tip) of the middle finger of your hand (on the same arm as the blood pressure
cuff). Use the Velcro strap to attach it firmly - neither loose nor tight.
4. Relax, put your hands in your lap and sit as still as possible to minimize any
movement artifact.
5. Click Start, the recorded pulse should look something like this.
6. Add a comment with the subject's name.
7. Inflate the cuff until the pressure is just above 180 mmHg. Note that the pulse
signal disappears.
8. Slowly deflate the cuff at a rate of 1 to 2 mmHg per second.
9. Once the pressure has reached 50 mmHg, completely deflate the blood pressure
cuff. Then click Stop.
Analysis (TABLE 5-3)

1. Examine your recording. Place the Waveform Cursor on the the first finger pulse
seen as the cuff pressure was falling. This represents the return of bloodflow to
the forearm.
2. Click on this point to enter the pressure in the Value panel and add the comment
"systolic pressure" to the data.
3. Drag the number from the value panel into the appropriate column of the table.

Exercise 4

This exercise is a variation on Exercise 3, with measurements taken from a different site
on the arm and with the arm in different positions.

Procedure

1. Wrap the cuff around the forearm, immediately above the wrist, of the same hand
which has the finger pulse transducer attached.
2. Ensure the subject's elbow is flexed at 90 degrees, with the wrist resting on a chair
arm or desk.
3. Type "arm resting, 90 " into the comment panel.
4. Click Start.
5. Inflate the cuff to 180 mmHg.
6. Press Add to enter the initial comment for this exercise.
7. Slowly deflate the cuff at a rate of 1 to 2 mmHg per second.
8. Once the pressure has reached 50 mmHg, completely deflate the blood pressure
cuff.
9. Click Stop.
10. Repeat steps 3 to 9, entering an appropriate descriptive comment each time, with
the arm in the following positions:
o Hanging down loosely by the side
o Held straight above the head.

Analysis (TABLE 5-4)

Determine the systolic blood pressure from the pressure cuff and finger pulse data.

1. Examine the finger pulse data. Place the Waveform Cursor on the the first pulse
following the reduction in cuff pressure. This represents the systolic pressure.
2. Click on this point to enter the pressure in the Value panel and add the comment
"systolic pressure" to the data.
3. Drag the number from the value panel into the appropriate column of the table.
4. Repeat steps 1-3 for each of the protocols in the exercise.
 EXERCISE VI

ECG & HEART SOUNDS

Introduction

The beating of the heart is associated with electrical activity and sound. The pattern of
electrical activity recorded at the body surface is called the electrocardiogram or ECG.
The aim of this laboratory is for you to record and analyze an ECG from a volunteer, and
to examine the relationship between the ECG and the characteristic sounds of the heart.

Learning Objectives

By the end of today's laboratory you will:

• Know where to place electrodes to record the standard limb lead ECG (leads I, II
and III )
• Be able to identify the major components of the ECG (P wave, QRS complex, T
wave) in these leads
• Be able to relate the electrical activity in the heart to these major components
• Be able to provide estimates of the timings of the components of the ECG and
their magnitudes
• Know how to calculate heart rate from the ECG
• Explain the time relationships between the electrical activity of the heart (as
recorded in the ECG) and the mechanical activity of the heart (as judged from the
heart sounds)

An early (1920s) cardiac

A teaching instrument microphone

Instruments for recording the activity of the heart.

Procedure

1. Make sure the PowerLab is connected and turned on.

2. Connect the push-button switch to Input 1
on the PowerLab.
3. Remove any watches and/or jewelry from
your wrists and ankles.
4. Connect the electrode lead wires to Earth,
CH1 NEG, and POS on the Bio Amp cable.
5. Plug the Bio Amp cable into the Bio Amp
input.

Standard Connection

Attach the positive electrode to the left wrist, the negative to the right wrist, and the
ground to the right leg.

1. Using a pen, mark each point where electrodes will be placed. Clean the skin with
alcohol swabs and lightly abrade the area with abrasive gel or a pad. This reduces
the electrical resistance of the outer layer of skin and ensures good electrical
contact.
2. If you are using the Reusable Clamp Electrodes, apply a small amount of
electrode cream to the electrodes before attaching. Electrode cream is not
necessary if you are using disposable electrodes which have electrode gel on them
already.
3. If, after looking at the signal during the first exercise, you find that this does not
produce a good signal, try the alternative method.
Exercise 1

You will record and examine the major components of the Electrocardiogram (ECG).

Procedure

1. The subject should relax and sit as still as possible to minimize signal artifacts due
to movements.
2. Type the subject's name into the Comment panel.
3. Click Start, then add the comment.

Click Autoscale as required to ensure that you can see all the data as it is being
recorded.

4. If the ECG cannot be seen, check that all three electrodes are correctly attached. If
the signal is noisy and indistinct, make sure that the subject is relaxed; consider
using the alternative attachment positions.
5. Click Stop.
6. Click Start again. While recording, ask the subject to open and close his or her
hands, and then move both arms across the chest.

The trace moves all over the place, and the ECG becomes distorted. This shows you
why it is necessary for subjects to keep still and stay relaxed while their ECG is being
recorded.

7. With the subject sitting quietly, click Start again. When you have a trace without
movement artifacts, type 'Resting ECG ' and the subject's name, and add the
comment.
8. Click Stop.
Analysis (TABLE 6-1)

1. Scroll through your data and observe the regularly occurring ECG cycles.
2. In a representative cycle, measure the amplitudes and durations of the P wave,
QRS complex and T wave.
3. To measure the amplitudes, place the Marker on the baseline immediately before
the P wave. Then move the Waveform Cursor to the peak of a wave. Click to
place the number in the Value panel.
4. Drag the number from the Value panel into the appropriate column of the upper
table.
5. To measure the durations, leave the Marker at the start of the wave or complex
and position the Waveform Cursor at the end of the wave or complex.
6. Click to place the number in the Value panel and then drag the number from the
Value panel into the appropriate column of the table.
7. Now investigate how the heart rate may vary from beat to beat. To do this, set the
horizontal compression to 10:1. Measure the time interval (in seconds) between
three pairs of adjacent R waves using the Marker and Waveform Cursor.
8. Record your results in the lower table. For each interval, the heart rate is shown in
column 3 of the table, calculated using the equation HR = 60 ÷ t , where HR =
Heart Rate (beats/min) and, t = time interval (seconds).
Study Questions

1. What can you say about the amplitude of the various waves in different cardiac
cycles?

2. The P wave and the QRS complex represent depolarization of the atrial and
ventricular muscle respectively. Why does the QRS complex have the largest
amplitude?

3. In steps 7 and 8 heart rate was calculated based upon the peak-to-peak interval of
the R waves. Was there variability between the beats? Would you expect the
interval between beats to be identical? Why or why not?

4. The range for a normal resting heart rate is 60 to 90 bpm. A trained athlete could
have a resting heart rate of 45 to 60 bpm. Why might a very fit person have a
slower heart rate than someone of average fitness?
Exercise 2

In this exercise, you will record the resting ECG signal from other members of your
group.

Procedure

1. Attach the electrodes to the new subject.

2. With the subject sitting quietly, click Start again. When you have a trace without
movement artifacts, type 'Resting ECG' and the subject's name and enter the
comment.
3. Click Stop.
4. Repeat for all other students in the group.

Analysis

Compare the duration and amplitude of the P waves, QRS complexes and T waves
between those in your group and with other members of the class. Record your results in
the Table. (TABLE 6-2)

To Measure Waveform Amplitude:

1. Drag the Marker to the lowest point of the waveform before each of the peaks of
interest.
2. Move the Waveform Cursor to the peak, to the right of the Marker, and click.
3. Drag the number from the ECG Value panel into the appropriate Amplitude
column of the table.

To Measure Waveform Duration:

1. Leave the Marker on the lowest point of the waveform before a peak.
2. Move the Waveform Cursor to the lowest point following the waveform, and
click.
3. Drag the number from the Time Value panel into the appropriate column of the
table.
Study Questions

1. Are the amplitudes and durations of the various waves in different individuals
similar or very different?

2. What variations in heart rate did you observe between individuals?

Exercise 3

You will measure and correlate the ECG and heart sounds in a resting volunteer.

Using a Stethoscope

The stethoscope bell is better than the diaphragm for this exercise because it blocks off
room noise. It still helps if everyone tries to keep the noise down.

The volunteer should place the bell of the stethoscope on the left side of their chest, using
the right hand. (It is easy enough to do this under one's shirt.) The stethoscope should be
moved to different positions until the student listening to the stethoscope hears clear heart
sounds. The sounds are soft, and room noise must be kept low. Once clear heart sounds
are heard, the volunteer should hold the stethoscope in place with the right hand while the
student listening to the stethoscope listens and records.

1. Click Start to record the ECG, and press the push-button switch on hearing 'lub'
and release it on 'dub'.
2. After a few heart beat cycles, click Stop.

Analysis

To make it easier to compare the recordings in the two channels, the LabTutor panel has
been set up to display the recordings overlaid. With the Channel Trace buttons you can
select which of the two channels is 'active' in the panel.

1. Note the correlation between Event and ECG signals.

2. Using the Marker and Waveform Cursor, follow the instructions below to
measure the time between the peak of the R wave and the Event signal going
high.
1. Select the ECG channel as active
2. Place the Marker on the R wave
3. Select the Event channel as active
4. Use the waveform cursor and select the Event signal going high
5. Insert this time into the table
3. Now measure the time between the peak of the T wave and the Event signal going
low.
1. Select the ECG channel as active
2. Place the Marker on the T wave
3. Select the Event channel as active
4. Use the waveform cursor and select the Event signal going low
5. Insert this time into the table

TABLE 6-3
Study Questions

1. Explain why ventricular contraction (systole) and the 'lub' sound occur
immediately after the QRS complex.

2. Explain why ventricular relaxation (diastole) and the 'dup' sound occur after the T
wave.
Exercise 4

You will record and correlate the ECG and heart sounds (with a cardiomicrophone) in a
resting volunteer.

Clearly, the method that you used in Exercise 3 is subject to considerable error. For
example, reaction time will introduce a significant delay.

The alternative, phonocardiography, is to use a microphone placed over the chest wall to
record the heart sounds which can then be displayed graphically in real time.

Procedure

1. Unplug the push button from Input 1 and plug the cardiomicrophone into Input 1.
2. Place the cardiomicrophone on the left side of your chest. Then hold it firmly in
place either by a strap running around the chest or by placing a heavy book or
similar object on top of it. (This requires that you lie down).

It is essential that the microphone is not held onto the chest-wall by hand, as the
inevitable movement of the hand introduces considerable noise into the recording.

3. Click Start to record the ECG and cardiomicrophone signals. You should try
placing the microphone in different positions to get the best possible signal.
4. After about 15 seconds, click Stop.

Analysis

Again, to make it easier to compare the recordings in the two channels, the LabTutor
panel is set up so that the recordings are overlaid.

Clicking the buttons towards the top right of the panel allows you to select which of the
two channels is 'active' in the panel.

1. Note the relationship between the R-wave and the first sound. Using the Marker
and Waveform Cursor, follow the instructions below to measure the time between
the peak of the R wave and the beginning of the first heart sound.
1. Select the ECG channel as active.
2. Place the Marker on the R wave.
3. Select the PCG channel as active.
4. Use the waveform cursor and select the beginning of the first heart sound.
5. Insert this time into the table.
2. Note the relationship between the T-wave and the second sound. Now measure
the time between the peak of the T wave and the beginning of the second heart
sound by repeating steps 1 to 5 above.

TABLE 6-4
Study Question

1. Your "lub-dub" recordings probably show some differences from the correct
timing of the heart sounds as judged by phonocardiography. How can you account
for this difference?
 EXERCISE VII

KIDNEY & URINE

Introduction

In this class, you will determine the approximate bladder capacity of an adult and revise
the structure and function of the kidneys. You will then perform urine tests on urine
samples representative of those obtained from four patients.

Learning Objectives

By the end of today's class you will be able to:

• State the approximate bladder capacity of a normal adult.

• Name the major anatomical features of the kidney.
• Correctly perform a dipstick test on urine samples.
• Give examples of diagnostic tests that can be performed on urine.
• Explain the importance of urinalysis and list some diseases that may affect urine
composition.
• Outline the difference between hemodialysis and peritoneal dialysis.
• Evaluate laboratory test data for a patient with a renal condition.
• Describe the counseling and training a kidney failure patient would require to
be prepared for home dialysis.

Exercise 1: Bladder Capacity

In this exercise you will determine the approximate bladder capacity of an adult.

Volunteer Preparation

This exercise requires three volunteers. Each will be asked to drink a liter of water and
delay urinating for as long as possible.

Establishing a good urine output depends on rapid absorption of the water so it is

important not to start with a full stomach. You should have eaten only a light meal and
should have avoided drinks containing caffeine in the 3–4 hours prior to this class.

Do not volunteer for this class if you are suffering from kidney or circulatory
problems, have any other medical problem, or are on any medications.
 Equipment

Ensure you have the following before starting the exercise:

• A large beaker or similar for collection of urine.

• A measuring flask.
• Paper towels for immediate clean up of any spills.
• Urine test strips.
• A watch or timer.

Procedure for Each Volunteer

Urine is a potentially infectious body fluid. Therefore, each volunteer is directly

responsible for measuring the volume of their own urine, and should clean up any spilled
urine themselves.

1. Enter your name and weight in the table.

2. Immediately drink a liter of water as quickly as possible.
3. Enter the time of drinking in the table.
4. Wait for as long as possible before emptying your bladder into the container provided.
(Note: Just before urinating, you should record your weight again.)
This exercise will be continued later in this class. Continue with the following
exercises in which you will revise kidney anatomy and learn how to use urine test strips.

Analysis – Bladder Capacity

Procedure

If you are ready to complete the bladder capacity exercise, please follow the instructions
below. If not, proceed to the next exercise and return to this page later.

1. Just before urinating, record your weight again.

2. Collect all of the urine. Measure the volume of urine and record it in the table.
3. Record your weight again.
Make sure that you are wearing exactly the same clothes each time you weigh
yourself.

TABLE 7-1
Study Questions

1. How much does a liter of water weigh?

2. What relationship did your weight changes have to your water intake and urine
volume? How are these relationships best explained?
Exercise 2: Kidney Anatomy

Each human kidney contains around one million functional units (nephrons). Let's make
sure we know the basic structure of the kidney.

Procedure

Write the names to the numbered structures in the diagram.

Exercise 3: Intravenous Pyelogram

In this exercise, you will identify structures in an intravenous pyelogram.

Mrs M, aged 57, has microscopic traces of blood in her urine. As part of her investigation
she had an intravenous pyelogram (IVP). One of the pictures taken during this procedure
is shown here. No abnormality was seen that could account for the blood in her urine.

Procedure

Write the names to the numbered structures in the image.

Intravenous pyelogram (IVP) of the urinary system.

 Exercise 4: Abdominal CT Scan

In many situations, computed tomography (CT) scans are replacing IVPs for kidney
investigations.

Write the names to the numbered structures in the image.

Abdominal CT scan viewed from below.

Procedure – Urine Testing

Analysis of blood and urine can be carried out by laboratories on request. However,
certain diagnostic tests can be performed quickly on urine using 'dipsticks' or urine test
strips. In this exercise you will learn how to correctly perform a dipstick test on a urine
sample. By the end of this exercise you should be able to describe how urine test strips
work and understand their limitations.

General Instructions on Using Urine Test Strips

• Check the expiry date on the bottle.

• Keep the bottle of strips closed except when withdrawing required items to prevent
contamination and to prevent reagents (chemicals) on the test strip reacting with
contaminants in the air.
• Be sure to have a color chart on hand BEFORE you begin the test.
• Avoid handling the actual test areas of the test strips.
• Be sure your hands, equipment and work area are clean before running tests.
 How to Use the Urine Test Strips

1. DIP: Completely immerse reagent areas of the strip in fresh urine and remove
immediately.
2. TAP: Gently tap the edge of the strip against the side of the container to remove excess
urine.
3. DAB: Dab or blot the back of the strip on a paper towel.
4. HOLD: Hold the strip in a horizontal position to prevent possible mixing of chemicals
from adjacent reagent areas. Start timer.
5. READ: At 30 seconds, start reading the strip by holding it over the matching color chart
on the bottle. Read bilirubin and glucose at exactly 30 seconds. Then read the others at
the times specified.

Exercise 6: Observation and Testing (Optional) of Urine

'Dipstick' testing of urine is carried out routinely in certain situations. For example, many
hospitals test the urines of all new patients, as do medical practices. During pregnancy
and in patients with a kidney disease it is used to monitor for the presence of protein in
the urine, which is an early sign of kidney injury.

In this exercise urine testing is optional. If you wish, you may just answer the
questions below.

Procedure

1. If you have obtained a urine sample for the bladder capacity exercise, you may choose to
test it using one of the urine test strips provided.
2. Use a testing strip to test your urine sample. Check with the test strip color chart.
Study Questions

1. Comment on the color of the urine samples. Why is the color of urine from some
students quite yellow and other students almost as pale as water?

2. Why do medical students need to know about the appearance of urine?

3. List some medical conditions in which a color change in the urine may be observed.
Analysis – Urine Observation and Testing

Some of the normal ranges for urine testing have been entered in the Table. (TABLE 7-2)

Examine these values and answer the questions:

1. Ketones in the urine are produced by breakdown of fatty acids. Under normal
conditions the body breaks down carbohydrates and uses glucose for fuel. Can
you name two conditions where ketones may be present in the urine?

2. 'Specific gravity' tests the concentration of the urine. A high specific gravity
indicates dehydration and possibly renal failure. Can you list two conditions, one
normal and one abnormal, that might be indicated by a low specific gravity?

3. List some medical conditions which might be indicated by the presence of blood
in the urine. [Hint: Look at Table 2 in the Background of this experiment for help]

4. Urine test strip measurements are semi-quantitative. Do you think these are as
accurate as the results that would be obtained by a hospital laboratory?
 EXERCISE VIII

WATER BALANCE

Introduction

In this experiment, you will investigate how the kidneys handle fluid loads. These include
water alone, and isosmotic salt and monosaccharide solutions, as well as a hyperosmotic
monosaccharide solution.

Learning Objectives

By the end of today's laboratory you will be able to:

• Describe how the kidneys handle a water load

• Distinguish between the handling of water and isosmotic salt loads
• Explain the pattern of fluid excretion following isosmotic and hyperosmotic
monosaccharide loads
• Discuss the relationship between urine osmolarity and specific gravity and how
osmolarity is affected by changes in urine flow rates

Claude Bernard (1813 - 1867) - the

Carl Ludwig (1816 - 1895) - a pioneer of renal
first to express the concept of the
physiology.
constancy of the internal environment.
Procedure

This laboratory involves the collection of urine at various time intervals and measurement
of its volume and specific gravity (an indication of osmolarity). There are four different
protocols, each requiring a different volunteer.

Volunteer preparation

The establishment of the diuresis depends upon fairly rapid absorption of the water so it
is important not to start with a full stomach. Just eat a light meal and drink normally in
the 3 to 4 hours before the laboratory starts. In addition, avoid fluids containing caffeine
(coffee, tea, cola drinks) for at least 3 hours prior to the laboratory.

Be sure to note the time at which you last urinated prior to coming to the laboratory.

Equipment

Please make sure you have the following equipment available before starting the
experiment.

1. A large beaker or similar container for collection of urine.

2. A measuring flask.
3. A Pasteur pippette for transfering a drop of urine to the refractometer.
4. A refractometer for measurement of urine specfic gravity.
5. Paper towels for the immediate cleaning up of any spills.

General procedures during the experiments for all volunteers

1. At the commencement of the experiment, note the time, collect your urine and
measure its volume. Keep a small sample for measurement of specific gravity.
2. Immediately after the collection of the first sample, drink the required solution
(except control). Once you have drunk this solution, do not drink anything else
during the laboratory.
3. Continue to collect urine approximately every 20 minutes, noting the time at
which the bladder is emptied to the nearest minute.
4. It will be found most convenient for each subject to be his or her own timekeeper;
there is no necessity for the subjects to keep in step with each other. The essential
thing is that the intervals between urination are accurately recorded.
Cautions

Do not volunteer to be a subject in this laboratory class if you are suffering from
kidney or circulatory problems, have any other medical problem or are on any
medications.

Urine is a potentially infectious body fluid. Therefore, students are directly

responsible for all measurements of the volume and specific gravity of their own urines,
and are required to clean up any spilt urine themselves.

Experimental protocols. There are four different protocols.

Protocol 1: Control - no fluid intake during experiment.

1. Drink nothing during this laboratory and collect specimens of urine each 20
minutes or so.
2. Measure the volume and specific gravity of your urine and then dispose of the
sample down the toilet.
3. Enter the volume and specific gravity of the sample into your own table.

Protocol 2: To illustrate a normal water diuresis.

1. Drink 800 mL of Solution 2, then collect specimens of urine each 20 minutes or

so.
2. Measure the volume and specific gravity of your urine and then dispose of the
sample down the toilet.
3. Enter the volume and specific gravity of the sample into your own table.

Protocol 3: To illustrate effects of drinking the equivalent of an isosmotic sodium

chloride solution.

1. Drink 800 mL of Solution 3, then collect specimens of urine each 20 minutes or

so.
2. Measure the volume and specific gravity of your urine and then dispose of the
sample down the toilet.
3. Enter the volume and specific gravity of the sample into your own table.

Protocol 4: To illustrate effects of drinking a hypersomotic glucose solution.

1. Drink 800 mL of Solution 4, then collect specimens of urine each 20 minutes or

so.
2. Measure the volume and specific gravity of your urine and then dispose of the
sample down the toilet.
3. Enter the volume and specific gravity of the sample into your own table.
Sample 1: 0 min

After collecting the samples complete the fields in the table below, for each different
protocol (Sample 1: 0 min). You must record the time in minutes that you last urinated
before you came to the laboratory.

Sample 2: 20 min

Sample 3: 40 min

Sample 4: 60 min

Sample 5: 80 min

Sample 6: 100 min

Sample 7: 120 min

Study Questions

While waiting to collect the samples, answer the following questions.

1. What changes in urine output do you expect to see in each of the protocols during
this laboratory?

2. What is the osmolarity of the fluid in the interstital space in the renal cortex? Is it
the same throughout that space?

3. What is the osmolarity of the fluid in the interstital space in the renal medulla? Is
it the same throughout that space?

4. What hormone is involved in regulating renal water excretion? What normally

inhibits the release of this hormone?

5. In question 4 you stated what normally inhibits the release of the hormone
controlling water excretion. From your own experience, identify two situations
where this inhibition is over-ridden. For each situation explain the physiologicial
advantange of over-riding this inhibition.
6. From your data collected so far, estimate the maximum volume of water that you
could drink over sixty minutes while still remaining in water balance?

7. What would happen if your water intake over sixty minutes significantly
exceeded the maximal volume you could excrete in this period? Why could this
be life threatening?

8. Provide a physiological explanation for the results for the isosmotic sodium
chloride solution and the hyposmotic solution (Protocol 3 - green vs Protocol 2 -
blue)?

9. Provide a physiological explanation for the results for the hyperosmotic glucose
solution and the hyposmotic solution (Protocol 4 - pink vs Protocol 2 - blue)?
Analysis 1: Flow and Osmolarity

Composite final results for flow and osmlolarity, for all four protocols, are shown in the
table. (TABLE 8-1)

10. Reflect on your predictions for the trends of each protocol in the spaces below.
Note that your original answers are shown but should not be changed.

Urine Flow and Specific Gravity Relationship

Show the graph with the scatterplot of urine flow in relationship to specific gravity for
your results.

Study Question

11. Explain the relationship, shown in the graph above, between the urine flow rate
and specific gravity.
 EXERCISE IX

GUT ABSORPTION

Introduction

In this experiment, you will investigate how the gut handles a carbohydrate load
presented as either glucose or starch.

Learning Objectives

By the end of today's laboratory you will be able to:

• Describe how the gut handles carbohydrates.

• Distinguish between the handling of simple and complex carbohydrates.
• Describe the hormonal control of blood glucose.

Frederick Banting (1891 - 1941)

and Charles Best (1899 - 1978) -
the discoverers of insulin, with one
of the first dogs they studied.

E. Waymouth Reid (1862 -

1948) - a pioneer in the study
of gut absorption.
Procedure

This laboratory involves the collection of finger prick blood samples at regular time
intervals and measurement of the blood glucose concentration. There are four different
protocols, each requiring a different volunteer.

Volunteer preparation

In this laboratory, it is important not to limit the rate at which the ingested fluid and
food reaches the intestine. Therefore, to avoid delays in gastric emptying, it is essential
that all subjects refrain from eating and drinking for at least two hours before starting this
laboratory!

Equipment

Please make sure you have the following equipment available before starting the
experiment.

1. A glucolet to obtain the blood samples.

2. A glucometer to measure the blood glucose concentration.
3. Alcohol wipes for disinfecting the finger prior to obtaining sample.
4. Paper towels to wipe any surfaces contaminated by blood.

General procedure for all volunteers

1. At the commencement of the experiment, obtain an initial blood sample.

2. Immediately after this, the volunteers (except control) must eat or drink the
required material. Once you have done this, do not eat or drink anything else
during the laboratory.
3. Continue to obtain blood samples every 15 minutes for the next 90 minutes.
4. It will be most convenient for each subject to be his or her own timekeeper. It is
essential that the intervals between samples are accurately recorded.

Cautions

Do not volunteer to be a subject in this laboratory class if you are suffering from any
medical problem or are on any medications.

Blood is a potentially infectious body fluid. Therefore, students are directly

responsible for all measurements on their own blood, and must ensure they clean any
surface contaminated with their blood.
Procedure

Blood is a potentially infectious body fluid. Therefore, students are directly

responsible for all measurements on their own blood, and must ensure that no surface is
contaminated with their blood.

Collecting a blood sample

To collect enough blood with each finger prick:

1. Use your non-dominant hand.

2. Ensure your hand is warm.
3. Have your hand below heart level.
4. Squeeze the finger from the hand to the tip.

Using the Glucolet

1. Clean the tip of a finger or thumb with an alcohol wipe.

2. Depending on the type of Glucolet you are using, follow the instructions of your
Lab Demonstrator on how to perform a finger prick.
3. Remember to place the used needle in the sharps disposal bin.

Measuring Blood Glucose Concentration

1. You will now need to measure the concentration of glucose in the blood;
2. This will be determined by the Glucometer you have available, your Lab
Demonstrator will show you how to measure this.

Measuring Blood Glucose Concentration

Experimental protocols

There are four different protocols illustrating the effects of different substrate absorption
in the gut. The table to the left indicates the amount to be ingested in each protocol and is
determined by the participants body weight.

Protocol 1: Control - no food or fluid intake.

1. Eat and drink nothing during this laboratory and collect blood samples each
15 minutes.
2. Measure the concentration of glucose in your sample.
3. Enter the concentration of glucose in your sample into your own table.

Protocol 2: Effects of drinking a glucose solution.

1. Drink the appropriate volume of glucose solution, then collect blood samples each
15 minutes.
2. Measure the concentration of glucose in your sample.
3. Enter the concentration of glucose in your sample into your own table.

Protocol 3: Effects of eating a complex carbohydrate.

1. Eat the appropriate amount of white bread, then collect blood samples each
15 minutes.
2. Measure the concentration of glucose in your sample.
3. Enter the concentration of glucose in your sample into your own table.

Protocol 4: Effects of eating a complex carbohydrate with fat.

1. Eat the appropriate amount of potato chips, then collect blood samples each
15 minutes.
2. Measure the concentration of glucose in your sample.
3. Enter the concentration of glucose in your sample into your own table.

After collecting the samples complete the fields in the table below, for each different
protocol (Sample 1: 0 min).

Sample 1: 0 min Sample 6: 75 min

Sample 2: 15 min Sample 7: 90 min

Sample 3: 30 min

Sample 4: 45 min

Sample 5: 60 min
Study Questions

While waiting to collect the next sample, answer the following questions.

1. What changes in blood glucose concentration do you expect to see in each of the
protocols during this laboratory?

2. In what regions of the body are carbohydrates digested? Of these, which region is
the most important?

3. What substances are absorbed from the stomach?

4. What factors influence the rate of gastric emptying?

5. What factors influence the rate of carbohydrate absorption from the small
intestine?
6. What cellular mechanisms are involved in glucose absorption from the small
intestine?

7. Describe what happens to the glucose that is absorbed from the gut.

8. Briefly describe how the liver handles a glucose load.

9. List the hormones that play a role in glucose metabolism. Of these, which is the
most important in regulating blood glucose after absorption of a glucose load?

10. Provide a physiological explanation for the blood glucose results of drinking the
glucose solution (Protocol 2 - blue) compared to ingesting the white bread
(Protocol 3 - green)?
 11. Provide a physiological explanation for the blood glucose results of ingesting the
white bread (Protocol 3 - green) compared to ingesting the potato chips
(Protocol 4 - pink)?

Analysis: Changes in blood glucose

Composite final results for all four protocols are shown in the table. (TABLE 9-1)

Study Question

12. Reflect on your predictions for each protocol in the spaces below. Note that your
original answers are shown.
 EXERCISE X

FROG SKELETAL MUSCLE

Introduction

In this laboratory, you will investigate the physiological properties of skeletal muscle
using the isolated frog gastrocnemius. Concepts that you will explore include the single
twitch, graded response and the relationship between muscle length and tension
generated. You will also explore tetanus and muscle fatigue. These experiments illustrate
the collective understanding of muscle physiology gained from over 400 years of
research.

Learning Objectives

By the end of today's laboratory you will be able to:

• Describe the relationship between the intensity of muscle stimulation and

contractile force.
• Describe and explain the effects of muscle stretch, summation, tetanus and fatigue
on the strength of contraction.

A drawing from the first documented use of the The myograph used by Etienne-Jules
isolated nerve-muscle preparation. This picture Marey in his study of skeletal
comes from a description by Jan Swammerdam muscle contraction, 'Du mouvement
(1637 - 1680) of his experiments in the 1660s. dans les fonctions de la vie ' (1868).

Dissection

You are required to dissect and mount the frog muscle yourself. Use the 'Previous' and
'Next' buttons to navigate through the dissection instructions.

Keep the tissue moist at all times with Frog Ringer's solution.
Exposing the Muscle

1. Follow the instructions on page 3 on obtaining a double-pithed frog.

2. Using a scalpel or sharp scissors, cut the skin of the frog around its abdomen.
3. Peel the skin down and off the legs of the frog.

Exposing the Gastrocnemius

1. Remove the leg from the frog by severing at the hip joint.
2. Carefully dissect the gastrocnemius muscle away from the tibio-fibula bone, but
leave it attached to the knee and the heel.
3. While the muscle is still attached pass a 15 cm piece of strong thread under the
Achilles tendon at the heel of the frog. Tie this thread securely to the tendon. You
will use this thread to attach the muscle to the force transducer.

Removing the Muscle

1. Sever the Achilles tendon below the attached thread.

2. Using bone shears or strong scissors, cut the tibio-fibula bone below the knee and
the femur bone above.
3. Keep the muscle in a Petri dish of cold Frog Ringer's solution until you are ready
to mount it in the muscle holder.
Equipment Setup

PowerLab

1. Connect the Bridge Pod to Input 1 of the PowerLab.

2. Connect the positive and negative BNC connectors of the stimulating electrode
leads to the analog Outputs on the PowerLab.

Force Transducer & Micropositioner

1. Mount the micropositioner on to the Retort Stand.

2. Securely mount the force transducer in the micropositioner mounting bracket.
3. Plug the force transducer cable into the back of the Bridge Pod.
Muscle Holder

1. Attach the Muscle Holder to the Retort Stand.

2. Fix the muscle in the Muscle Holder by positioning the knee joint just below the
constriction in the perspex molding, as shown in the figure.
3. Connect the alligator clips from the stimulating electrodes to the inner two
connectors at the top of the muscle holder.

Equipment Setup

Retort Stand

1. Secure the thread attached to the achilles tendon of the muscle through the hole in
the metal tab of the force transducer. Make sure that there is some slack in the
thread.
2. Raise the micropositioner using the adjustment knob so that the muscle is vertical
but not under tension. The thread should not be loose, but should have some slack
in it. Make sure that there is room to increase the height of the force transducer by
at least 10 mm.
3. Check that the stimulating electrodes on the muscle holder are in contact with the
muscle
4. Rotate the base of the muscle holder and insert an appropriately sized beaker to
collect waste solution.
5. Finally, rinse the muscle with Frog Ringer's solution to keep it moist.
Force Transducer Setup

Raw output from the force transducer is in millivolts. It needs to be calibrated to give the
more meaningful units of Newtons (N). Force transducers also often have some residual
offset voltage that needs to be corrected for.

Zeroing Procedure

1. Leave the force transducer undisturbed and click Start.

2. The baseline value will be displayed in the LabTutor panel. Use the knob on the
front of the Bridge Pod to adjust the baseline value to zero, then Click Stop.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp'
link to correct for any residual offset voltage.

Calibrating Procedure

1. Once the force transducer has been zeroed, click Start and record for about 5
seconds, then click Stop.
2. Now hang a known weight (between 5 and 50 grams) from the force transducer
and record for another
5 seconds, then click Stop.
3. Enter the weight in the Unit Conversion table to the left and LabTutor will
calculate the N value.
4. Use the Waveform Cursor to select a stable section of the record before you added
the weight, then click the arrow next to 'Point 1' in the Calibration Panel and, if it
is not already entered, type '0' in the right-hand box.
5. Now select a stable section of the record after you added the weight, click the
arrow next to 'Point 2' and enter the calculated N value in the right-hand box.
6. Click the 'Apply' button to implement the calibration.

Exercise 1

In this exercise, you will progressively increase the strength of stimulation and record the
effects on the force of muscle contraction.

The LabTutor panel is displaying data using Record Overlay.

It is essential that throughout this Laboratory you watch what is actually happening to
the muscle. You will be asked to describe your observations and these descriptions will
be included in your Report.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link to
correct for any residual offset voltage again.
Procedure

1. Make sure the muscle is moist and is in contact with the stimulating electrodes.
2. First, determine the threshold for the preparation using a range of stimulus
amplitudes. In the Stimulator panel check that the voltage is set to 50 mV and the
number of pulses is set to 1 (with 1 pulse, stimulus interval is irrelevant).
3. Click Start. The software is set up so that LabTutor will now record for 625 ms
with a single stimulus being delivered after an initial delay of 50 ms.
4. The contractile response of the muscle and the time at which the stimulus was
delivered are shown in the LabTutor panel. LabTutor will also enter a comment
showing the amplitude of stimulation.
5. Continue to increase the stimulus amplitude in 50 mV steps, recording the
responses until you have 3 successive stimuli that do not produce any further
response.

Analysis

You will have a number of records to analyze. Analyze all those that show a measurable
response, plus the record immediately before the first to show a response (enter a force of
zero for this record). You should analyze them in ascending order of stimulus amplitude.

Procedure

For each record that gave a response:

1. Stimulus Voltage:
o In the Stimulus vs Force table, enter the stimulus amplitude displayed in
the Comment for the recording.
2. Twitch response:
o Place the Marker on the baseline recording prior to the beginning of the
stimulus. Move the Wavefrom Cursor over the recording and click on the
peak of the response to transfer its value to the Value panel.
o Now drag the Force value from the Value panel to the appropriate cell in
the 'Force (N)' column of the table.

As you enter the data, the Graph panel will graphically display the relationship between
stimulus voltage and response size.

Note the stimulus voltage at which the response no longer increases. This is called the
'Maximum Excitation Voltage'. To determine your value for a supramaximal stimulus,
enter this voltage in the Supramaximal Excitation table. The voltage that you enter is
multiplied by 1.5 to obtain the supramaximal excitation voltage, shown in the second
column.
Study Questions

1. What was the smallest voltage required to produce a contraction (the threshold
voltage)? What proportion of the fibers in the muscle do you think were
contracting to produce this small response?

2. What was the smallest voltage required to produce the maximum (largest)
contraction? What proportion of the fibers in the muscle do you think were
contracting to produce this maximal response?

3. What do you conclude happened to the number of fibers contracting as the

voltage was raised from threshold to that required to produce a maximal
contraction?

4. In light of the all or none law of muscle contraction, how can you explain the
graded response?
Exercise 2

In this experiment, you will progressively increase the stretch on the muscle and record
the effects on the force of muscle contraction.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link
to correct for any residual offset voltage again.

Procedure

1. Make sure the muscle is moist and is in contact with the stimulating electrodes.
2. The Micropositioner should be adjusted so that the thread is not slack, nor under
tension.
3. Note the position of the Micropositioner. This is your reference position.
4. In the Stimulator panel, insert your value for the supramaximal stimulus voltage
that you calculated in Exercise 1.
5. Check that the number of pulses is set to 1.
6. Click Start. The software is set up so that LabTutor will now record for 625 ms
with a single stimulus being delivered after an initial delay of 50 ms.

The contractile response of the muscle and the time at which the stimulus was
delivered are shown in the LabTutor panel. LabTutor automatically adds a
comment at the time of stimulation.

7. Edit the comment to show the current length

(eg; 'Length = 0' for your first record).
8. Raise the micropositioner by one millimeter by turning the adjustment knob
located on the top of it. Do not try to reposition the entire unit on the ring stand.
9. Wait at least 10 seconds to allow the muscle to recover.
10. Repeat steps 6 to 9 for every 1 mm increase in height, entering appropriate length
comments for each adjustment, until you have increased the height by a total of
10 mm.
11. At the end of the recording, return the muscle to its original length by turning the
adjustment knob on the micropositioner.
Analysis

You will have eleven records to analyze.

1. Using the Waveform Cursor and the Value panel, measure the baseline force
value. This is the resting or preload force.
2. Next, determine the twitch force by placing the cursor on the maximum value in
the record. Enter your results in the Table.

The last column of the table shows the net force obtained by subtracting the resting
(preload) value from the twitch force value.

3. Repeat steps 1 and 2 for the remaining 10 records. Note that as the muscle is
stretched the resting value is increasing progressively.

As you enter the data, the Plot panel will graphically display the relationship between
stretch and force of contraction. It will show the resting force, the twitch force and the net
force.

Study Questions

1. What effect does stretching the muscle have on contraction strength? Is this effect
linear?

2. What stretch resulted in the highest contraction force? What happens to the
muscle at the highest stretch levels?
Exercise 3

In this exercise, you will stimulate the muscle with twin pulses at different pulse intervals
and observe the effect on muscle contractions.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link
to correct for any residual offset voltage again.

Procedure

1. Make sure the muscle is moist and is in contact with the stimulating electrodes.
2. In the Stimulator panel, insert your value for the supramaximal stimulus voltage
that you calculated in Exercise 1.
3. Set the Stimulator Panel to deliver 2 pulses. Check that the interval between the
two pulses is set to 400 ms (a frequency of 2.5 Hz)
4. Click Start. LabTutor is set to record for 625 ms. It will deliver the first
supramaximal stimulus just after you click Start, and the second after the interval
that you specify. LabTutor will also enter a comment showing the stimulus
amplitude.
5. Repeat step 4 decreasing the stimulus interval in the following sequence: 200 ms,
100 ms, 80 ms, 60 ms, 40 ms, 20 ms.

Analysis

You will have seven records to analyze.

1. For each record, place the Marker on the baseline immediately before the first
contraction. Then use the Waveform Cursor to measure the maximum heights of
the two contractions
2. Click on each peak to transfer the value into the Value panel.
3. Drag the value to the appropriate place in the Table.

When the peaks merge, put the single measurement in the 'Force (2nd response)'
column of the table.

Study Question

1. How does varying the frequency effect contraction force? Which interval caused
the greatest contraction?
Exercise 4

Here you will examine the muscle's response to a series of stimuli at different
frequencies.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link
to correct for any residual offset voltage again.

Procedure

1. Make sure the muscle is moist and is in contact with the stimulating electrodes.
2. In the Stimulator panel, insert your value for the supramaximal stimulus voltage
that you calculated in Exercise 1.
3. Set the Stimulator Panel to deliver 60 pulses. Check that the interval between
pulses is set to 400 ms.
4. Click Start. LabTutor will record for 1250 ms. It will deliver the first
supramaximal stimulus 50 ms after you click Start. LabTutor will also enter a
comment showing the stimulus amplitude.
5. Repeat step 4 decreasing the stimulus interval in the following sequence: 200 ms,
100 ms, 80 ms, 60 ms, 40 ms, 20 ms.

Analysis

You will have seven records to analyze.

1. For each record, place the Marker on the baseline immediately before stimulation
begins. Use the Waveform Cursor to measure the maximum force of contraction
for the record.
2. Click to transfer the value into the Value panel.
3. Drag the value to the appropriate place in the Table.

Where the contractions are distinct, measure the one greatest in amplitude.

Study Question

1. Define tetanus. At which stimulus interval did you observe tetanus? Explain the
mechanism behind this phenomenon.
Exercise 5

Here you will examine the muscle's response to a sustained series of stimuli at a high
frequency.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link
to correct for any residual offset voltage again.

Procedure

1. Make sure the muscle is moist and is in contact with the stimulating electrodes.
2. In the Stimulator panel, insert your value for the supramaximal stimulus voltage
that you calculated in Exercise 1.
3. Set the Interval between pulses to 20 ms and the number of pulses to 1750.
4. Click Start. After a 50 ms delay LabTutor will stimulate the muscle for 35
seconds with pulses at 20 ms intervals. Recording will continue for a further 10
seconds after the last stimulus.

Analysis

You will have a single record to analyze.

1. Place the Marker on the baseline immediately before the start of stimulation.
2. Position the Waveform Cursor at the time of maximal force generation. Click to
place the Δ Force value in the Value panel then drag it to the appropriate cell in
the table.
3. Now measure the force when it has declined to a minimum, immediately prior to
the end of stimulation, and enter the value in the appropriate cell of the table.

The last column of the table shows the percentage decline in contraction force from
maximum to minimum.
Study Questions

1. At what time point did your muscle begin to fatigue? Comment on the percentage
decrease in contraction force by the end of the experiment.

2. Provide a possible mechanism for why the muscle was unable to maintain a
prolonged contraction.

3. Would your results have differed if you were measuring from smooth muscle
tissue? Why?
 EXERCISE XI

FROG NEUROMUSCULAR JUNCTION

Introduction

In this laboratory, you will investigate the physiological relationship between skeletal
muscle and motor nerve supply. Using an isolated frog gastrocnemius (calf muscle) and
sciatic nerve you will explore twitch recruitment, muscle fatigue and investigate the
effects of tubocurarine (a muscle relaxant).

A drawing from the first documented use of the isolated nerve-muscle preparation.
This picture comes from a description by Jan Swammerdam in the 1660s.

Learning Objectives

By the end of today's laboratory, you will be able to:

• Describe the relationship between the intensity of nerve stimulation and contractile
force.
• Describe the effect of fatigue on the strength of contraction.
• Explain the effect of tubocurarine on nerve and muscle function.

Dissection

You are required to dissect and mount your frog nerve. Use the 'Previous' and 'Next'
buttons to navigate through the dissection instructions.

Gross Dissection

1. Obtain a double-pithed frog following the instructions in page 3. Secure the

animal ventral side up to a dissecting board using straight pins.
2. Place the frog in a dissection pan, and keep the animal moist at all times with
Frog Ringer's solution.
3. Using a scalpel or sharp scissors, cut the skin of the frog around its abdomen.
4. Peel the skin down and off the legs of the frog.
5. Keep the tissue moist at all times with Frog Ringer's solution.
Exposing the Sciatic Nerve

1. Grasp the urostyle with forceps and cut it free; you should be able to observe the
nerve plexus below it. Be careful not to damage the nerve plexus.
2. Using a glass hook, locate and lift the sciatic nerve free from the associated fascia
and the sciatic artery. You may need to use blunt dissection techniques.
3. Cut the nerve from the spinal cord and reflect the nerve back onto the animal's
leg.
4. Tie a piece of thread around the free end of the nerve so that it can be handled
gently.

Removing the Muscle

1. Sever the Achilles tendon below the attached thread.

2. Using bone shears or strong scissors, cut the tibio-fibula bone below the knee and
the femur bone above.
3. Keep the muscle/nerve preparation in a Petri dish of cold Frog Ringer's solution
until you are ready to mount it in the muscle holder.
Equipment Setup

PowerLab

1. Connect the Bridge Pod to the pod port on Input 1 of the PowerLab.
2. Connect the positive and negative BNC connectors of the stimulating electrode
leads to the analog outputs on the PowerLab.

Micropositioner

1. Securely mount the force transducer in the Micropositioner mounting bracket.

2. Plug the cable into the back of the Bridge Pod.

Muscle Holder

1. Attach the muscle holder to the retort stand.

2. Fix the muscle and nerve in the muscle holder by positioning the knee joint just
below the constriction in the perspex molding. The gastrocnemus should be oriented
up and the sciatic nerve gently draped over the stimulator leads.

Be careful not to handle the sciatic nerve with metal instruments.

3. Rotate the base of the muscle holder and insert an appropriately sized beaker.
4. Rinse the muscle with Ringer’s solution to keep it moist.
Retort Stand

1. Secure the thread attached to the Achilles tendon of the muscle through the hole in
the metal tab of the force transducer. Make sure that there is some slack in the
thread.
2. Raise the Micropositioner using the adjustment knob so that the muscle is vertical
but not under tension. The thread should not be loose, but should have some slack in
it. Ensure that there is room to increase the height of the force transducer by at least
10 mm.
3. Connect the alligator clips from the stimulating electrodes to the outer two
connectors at the top of the muscle holder.
4. Check that the stimulating electrodes on the muscle holder are in contact with the
nerve.

Force Transducer Setup

Raw output from the force transducer is in millivolts. This needs to be calibrated to give
the more meaningful units of Newtons (N). Force transducers also often have some
residual offset voltage that needs to be corrected for.

Zeroing Procedure

1. Leave the force transducer undisturbed and click Start.

2. The baseline value will be displayed in the LabTutor panel. Use the knob on the
front of the Bridge pod to adjust the baseline value to zero, then click Stop.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp'
laink to correct for any residual offset voltage.
Calibrating Procedure

1. Once the force transducer has been zeroed, click Start and record for about 5
seconds, then click Stop.
2. Now hang a known weight (between 5 and 50 grams) from the force transducer and
record for another
5 seconds, then click Stop.
3. Enter the weight in the Unit Conversion table to calculate the force value (N).
4. Use the Waveform Cursor to select a stable section of the record before you added
the weight, then click the arrow next to 'Point 1' in the Calibration Panel and, if it is
not already entered, enter '0' in the right-hand box.
5. Now select a stable section of the record with the weight added. Click the arrow
next to 'Point 2' and enter the calculated N value in the right-hand box.
6. Click the 'Apply' button to implement the calibration.

Exercise 1: Twitch Recruitment

In this exercise, you will progressively increase the strength of nerve or muscle
stimulation and record the effects on the force of muscle contraction.

It is essential that throughout this laboratory you watch what is actually happening to
the muscle. You will be asked to describe your observations.

Procedure - Nerve Stimulation

The LabTutor panel is displaying data using Record Overlay.

1. Make sure the muscle and nerve are moist and the nerve is in contact with the
stimulating electrodes.
2. If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp'
link to correct any residual offset voltage.
3. Ensure the number of pulses is set to 1 in the Stimulator panel.
4. Click Start. The software is set up so that LabTutor will now record for 625 ms with
a single stimulus being delivered after an initial delay of 50 ms.
5. The contractile response of the muscle and the time at which the stimulus was
delivered are shown in the LabTutor panel. LabTutor will also enter a comment
showing the amplitude of stimulation. Edit this comment to also say 'nerve
stimulation'.
6. First, you should determine the threshold for the preparation using a range of
stimulus amplitudes in the Stimulator panel.
7. Once you have found the threshold (typically ~50mV), increase the stimulus
amplitude in 5 mV steps, recording the responses until you have three successive
stimuli that do not produce any further increase in the amplitude of the muscle
contraction.
Exercise 1: Twitch Recruitment

Procedure - Muscle Stimulation

Now you will stimulate the muscle directly.

1. Move the stimulating electrode leads to the electrodes in contact with the muscle on
the Muscle Holder.
2. Make sure the muscle and nerve are still moist and the muscle is in contact with the
stimulating electrodes.
3. If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp'
link to correct any residual offset voltage.
4. Again, using the Stimulator panel ensure the number of pulses is set to 1 and the
stimulus amplitude is set to 50 mV.
5. Click Start and change the resulting comment to include 'muscle stimulation'.
6. You should determine the threshold for the preparation using a range of stimulus
amplitudes in the Stimulator panel.
7. Once you have found the threshold, increase the stimulus amplitude in 50 mV
steps, recording the responses in the LabTutor panel until you have three successive
stimuli that do not produce any further increase in the amplitude of the muscle
contraction.

Analysis: Twitch Force

This analysis will also determine the voltage at which you should stimulate either the
nerve or muscle for the remainder of the experiment ― "Supramaximal Excitation
Voltage".

You will have a number of records to analyze for both stimulation of the nerve and
stimulation of the muscle. You should analyze them in ascending order of stimulus
amplitude.

Procedure

For the record immediately before the first to show a response, and each subsequent
stimulus voltage that produced a response:

1. From the LabTutor Panel enter the stimulus amplitude displayed in the
Comment into the appropriate Stimulus vs Force table.
2. Place the Marker on the baseline recording prior to the beginning of the stimulus.
Move the Waveform Cursor over the recording and click on the peak of the
response to display its value in the Value panel.
3. Drag the Force value from the Value panel to the appropriate cell in the 'Force (N)'
column of the table.
4. Continue this analysis for all appropriate nerve and muscle stimulation records.
Analysis: Excitation Voltage

The Supramaximal Excitation Voltage table shows the voltage that gave the maximum
response for nerve and muscle stimulation.

The stimulus voltage at which the response no longer increases is called the Maximum
Excitation Voltage.

The maximum excitation voltage is multiplied by 1.5 to obtain the Supramaximal

Excitation Voltage. This is the voltage at which you should stimulate either the nerve or
muscle for the remainder of the experiment

Analysis: Twitch Latency

Now you will compare the time it took for the muscle to respond to either nerve
stimulation or direct muscle stimulation.

1. Select a record that shows a good strong response to nerve stimulation.

2. Place the Marker at the beginning of the stimulus.
3. Move the Waveform Cursor over the recording and click at the start of the twitch
response to display the value in the Value panel.
4. Now drag the Time value from the Value panel to the appropriate cell in the table.
5. Move the Waveform Cursor over the recording and click at the top of the twitch
response to display the value in the Value panel.
6. Again, drag the Time value from the Value panel to the appropriate cell in the table.
7. Select a record that shows a strong response to muscle stimulation and repeat steps
two to six above.

Exercise 2: Fatigue

In this exercise, you will examine the muscle's response to a sustained series of stimuli at
a high frequency.

If you are using the ADInstruments Bridge Amp please use the 'Zero Bridge Amp' link
to correct any residual offset voltage.

Procedure - Muscle

1. You should be stimulating the muscle directly, so check that the connections on the
Muscle Holder are appropriate.
2. In the Stimulator panel, insert your value for the supramaximal muscle stimulus
voltage that was calculated in Exercise 1.
3. Set the Interval between pulses to 20 ms and the number of pulses to 1500.
4. Make sure the muscle and nerve are moist.
5. Click Start. LabTutor will stimulate the muscle for 30 s with pulses at 20 ms
intervals. Recording will continue for a further 10 s after the last stimulus.
Procedure - Nerve

Wait 10 minutes before stimulating the nerve directly.

1. Change the connections on the Muscle Holder to now stimulate the nerve.
2. Make sure the muscle and nerve are moist.
3. In the Stimulator panel, insert your value for the supramaximal nerve stimulus
voltage that was calculated in Exercise 1.
4. Set the Interval between pulses to 20 ms and the number of pulses to 1500.
5. Click Start and record as before.

Analysis: Fatigue

You will have a two records to analyze: one for nerve stimulation and one for direct
muscle stimulation.

1. Place the Marker on the baseline immediately before the start of stimulation.
2. Use the Waveform Cursor to measure the contraction force at times of 1, 5, 10, 15,
20, 25, and 30 seconds after the start of the stimulation.
3. Click on the waveform at each of these times to display the value in the Value panel.
You can switch between muscle and nerve records using the record overlay buttons.
4. Drag the values to the appropriate cells in the table.
5. The Muscle Fatigue Summary table will calculate the the change in contraction
force at the end of the stimulus as a percentage of the maximal force.

Exercise 3: Locating the site of action of tubocurarine

In this exercise, you will continuously stimulate the nerve at a low frequency, then add
tubocurarine and examine the effect.

Procedure

Nerve Stimulation, Tubocurarine on Nerve

1. Make sure the muscle and nerve are moist and the nerve is in contact with the
stimulating electrodes.
2. In the Stimulator panel, enter the supramaximal nerve stimulus voltage that you
calculated in Exercise 1.
3. Set the Interval between pulses to 10000 ms and the number of pulses to 1500.
4. Click Start. LabTutor will stimulate the nerve once every 10 s.
5. Apply tubocurarine (10 µM) directly to the nerve. The LabTutor panel will add a
Comment showing the amplitude of the stimulus, change this to include 'nerve
stimulation, nerve'.
6. Click Stop after 10 minutes.
Nerve Stimulation, Tubocurarine on Muscle

1. Check that the stimulus setting are the same as above.

2. Click Start. LabTutor will stimulate the nerve once every 10 s.
3. Apply tubocurarine (10 µM) directly to the muscle and edit the comment to include
'nerve stimulation, muscle'.
4. Click Stop after 10 minutes.

Muscle stimulation, Tubocurarine on Muscle

1. Move the stimulating electrode leads to the electrodes in contact with the muscle on
the Muscle Holder.
2. In the Stimulator panel, enter the supramaximal muscle stimulus voltage that you
calculated in Exercise 1.
3. Check that the other stimulus settings are the same as above.
4. Click Start. LabTutor will stimulate the muscle once every 10 s.
5. Apply tubocurarine (10 µM) directly to the muscle and edit the comment to include
'muscle stimulation, muscle'.
6. Click Stop after 10 minutes.

Describe the effects of tubocurarine in the answer box.

 EXERCISE XII

REFLEXES

Introduction

In this laboratory, you will investigate your reflexes in response to a variety of stimuli
and under a variety of conditions. You will examine some simple and complex reflexes
from a volunteer.

Learning Objectives

By the end of today's laboratory you will be able to :

• Elicit the axon reflex

• Elicit the different tendon reflexes
• Elicit pupillary and other eye reflexes
• Elicit an example of a flexion withdrawal reflex

A diagram, from
Sherrington's famous
Figure 7 of De Homine (1662), by René Descartes (1594- book,
1660), illustrates his concept of what is involved in a The Integrative Action
reflex response. The heat of the fire causes movements of of the Nervous System
animal spirits in hollow nerves. These open pores in the (first published in
brain that result in spirits inflating the leg muscles so that 1906),
the leg is removed from the source of the heat. showing components
of reflex behaviour.
Exercise 1

In this exercise, you will observe an axon reflex by scratching the skin.

Procedure

Scratch the skin of the forearm firmly with a blunt and rough instrument such as a probe
and observe the response.

Analysis

1. Note the initial and the late appearance of the scratched area.

2. Record the time required for the development of the red flare and measure the
width in mm 5, 10 and 15 minutes after the skin was scratched.

Study Question

1. Describe the response of the skin to scratching (Triple Response) and explain the
mechanisms of the responses.
Exercise 2

In this exercise, you will observe a myotatic reflex.

Patellar Tendon Reflex

Procedure

1. Ask the volunteer to sit in a chair and cross the leg over the other leg, allowing the
foot to swing backwards and forwards freely.
2. With the Tendon Hammer, tap the patellar tendon just below the knee to elicit the
knee jerk response. Practice this exercise a few times so that you are comfortable
in getting a reliable response. The appropriate response is contraction of the
quadriceps femoris muscle, which causes extension of the crossed leg.
3. Now ask the volunteer to perform the 'Jendrassik maneuver'. To do this, cup and
link the fingers of both hands, and then pull strongly outwards across their chest.
4. While the volunteer performs this maneuver, use the Tendon Hammer to tap the
patellar tendon.

Analysis

1. You will analyze and compare the latency and change in leg angle under normal
conditions and while performing the Jendrassik maneuver.

Study Question

1. Describe the difference in the responses of the leg under normal conditions and
with Jendrassik maneuver while performing the Knee Jerk Reflex.

Achilles Tendon Reflex

Procedure

1. Ask a volunteer to kneel across the bottom of a chair with the feet hanging in a
relaxed state over the edge.

2. With the Tendon Hammer, tap the Achilles tendon to elicit a response. The
appropriate response is contraction of the gastrocnemius and soleus to produce
plantar flexion.
Biceps Reflex

Procedure

1. Ask a volunteer to flex his/her forearm to 90° supported by the examiner’s arm.

2. The examiner palpates with the thumb of the supporting arm for the volunteer’s
biceps tendon. When it is located, the thumb remains in position over the tendon,
and the examiner’s thumb is tapped with the tendon hammer. The appropriate
response is contraction of the biceps muscle which flexes the arm of the volunteer.

Study Questions

1. Discuss the mechanism of the tendon reflexes taking into account the
components of the reflex arc.

Exercise 3
The retina of the eye is able to respond to differences in light intensity over a very wide
range. In bright light, the eye's sensitivity is low, but in dark conditions the sensitivity
increases. Most of this adaptation occurs in the photoreceptor cells of the retina, but part
of it results from regulation of the amount of light entering the eye through the pupil.

Pupillary Reflexes

Procedure

1. Shade the volunteer's eyes for about 15 seconds.

2. Shine a light into one eye and note the response.

What is the response of the pupil when light is shone on it?

3. Repeat steps 1 and 2, but note the response of the pupil in the unstimulated eye.

What was the response of the pupil in the opposite eye?

4. With normal lighting, ask the subject to look into the distance and then at an
object held close up (about 10 cm from the eye).

What happens to pupil diameter when the eye is focused for near vision?

Blink Reflex
Procedure

Pass a hand very rapidly in front of the volunteer’s face and observe the response.
Do not touch the eye and avoid stirring up air currents which may stimulate
conjunctival or corneal reflexes.

Study Question

1. What is the response of the eyes upon eliciting the blink reflex? Discuss its
mechanism.

Exercise 3
The flexion withdrawal reflex is not readily studied in human volunteers, because an
unpleasantly painful stimulus is needed to evoke it. However, a little-known reflex
involving an obscure muscle in the hand, the palmaris brevis muscle, exists that shares
some features of the flexion reflex and is easily evoked. Everyone in the group should try
this exercise.

Procedure

1. Cup your hand. Note a dimpling of the skin along the ulnar border (figure). This
dimpling is the action of the palmaris brevis muscle.

In some people, it is difficult to see the dimpling of the skin; reflex effects will then
be hard to demonstrate.

2. With the palm of your hand facing up and the hand relaxed, press with your
fingernail over the pisiform bone (figure). Firm pressure causes a reflex
contraction of the palmaris brevis muscle.
3. Try to find other bony prominences in the hand at which the reflex can be elicited.
4. Attempt to contract the palmaris brevis muscle voluntarily, without moving your
little finger. Most people find this difficult or impossible. However, with
extensive practice the movement can be learned.

Palmar view of the left hand, showing where the palmaris brevis
muscle dimples the skin and the location of the pisiform bone.

EXERCISE XIII
 SENSORY PHYSIOLOGY

Introduction

Senses are the physiological methods of perception. Aristotle was first to classify them
into the five senses you are most familiar with: sight, hearing, taste, smell and touch.
There are at least four other senses: body awareness, balance, heat and pain. Other
organisms have senses not possessed by humans. In this laboratory you will become
familiar with and make observations based on your own senses.

Learning Objectives

By the end of today's laboratory you will have:

• Observed various aspects of vision, including the blind spot, after-images and
some illusions
• Determined the distribution of touch receptors in different parts of the body, as
determined by two-point discrimination
• Demonstrated that the sensation of taste is strongly influenced by smell and
investigated the distribution of taste receptors on the tongue
• Demonstrated the joint position sense
• Evaluated the role of the semicircular canals in determining the position of the
head in space

Les cinq sens (The five senses) by Louis-Leopold Boilly (1761-1845)

Reproduced courtesy of the National Library of Medicine (USA).

Exercise 1: Convergence of Gaze

Binocular vision requires that the separate images in the right and left eyes be "fused" to
give a single view. Fusion of the images of an object is possible only if the images fall on
corresponding parts of the right and left retinae. If they do not, a double view of the
object results.

Procedure

1. Ask a volunteer to look first at a distant object, and then at an object held close up
(15 cm from the face). Note that the volunteer's eyes are turned inwards when
looking at a close object.
2. Hold one arm outstretched, with the index finger upright and in line with some
distant object (for example a clock on a far wall). Look at your finger (keep it in
focus), but concentrate your attention on the distant object. Note that the distant
object is seen doubled: there are two images, side by side.
3. Cover the right eye. Note that the right image of the distant object disappears.
4. With both eyes open, look at the distant object. Note that your finger is seen
doubled.
5. Cover the right eye. Note that the left image of your finger disappears.
6. Place your fingers as shown and hold your arms out from your body. With both
eyes open, look at the distant object. Note that the tips of your fingers seem to
float in space.

Describe what you saw when you performed the convergence of gaze exercise.

Figure 1: Index finger in line of sight but observing a distant object

Figure 2: Distant object in line of sight but

observing index finger

Exercise 2: Accommodation (Focusing)

The eye can accommodate (change focus) for far or near vision, by varying the shape of
the lens.

Procedure

1. Cover or close one eye, and hold a pin about 15 cm in front of the other eye, in
line with some distant object.
2. Look at the distant object and note that the pin appears blurred and dim: it is out
of focus.
3. Now look at the pin. Note that the distant object becomes dim and indistinct. Note
also that accommodation for the near object (the pin) is accompanied by a feeling
of effort.
4. If you are wearing glasses, remove them now. Cover one eye and hold the pin at
arm's length. While looking at the point of the pin, slowly bring it toward the face
until it becomes blurred. The shortest distance at which the pin can be kept in
focus is the 'near point'. Measure this distance with a ruler and insert the value in
the table, noting whether or not you are nearsighted (myopic).

Exercise 3: Saccadic masking

Can you see your own eyes moving?

Procedure

1. Look into a mirror 30 to 60 cm away.

2. Alternately look directly at the image of your right eye and then your left eye.
3. Did you detect any movement of your eyes?
4. Have another member of your group look at your eyes while you are doing this.
5. Did he or she detect any movement of your eyes?

Exercise 4: The Blind Spot

The visual field for each eye includes a blind spot, representing the optic disc - a part of
the retina with no photoreceptors.

Procedure

1. Obtain a felt-tipped pen that writes with black ink but has a white barrel.
Alternatively, wrap some white paper around the barrel of a black fiber-tipped
pen, leaving only the black writing tip exposed.
2. Mark a small X on a piece of white paper. Close the left eye and look steadily at
the cross, at a distance of about 25 cm. For the rest of this exercise, keep your
head completely still and continue to look at the cross.
3. Move the pen out (to the right) from the cross. At a certain distance the tip will
become invisible. Mark this place with a spot on the paper.
 4. Carry the pen further to the right, until it becomes visible again. Mark this place
with another spot.
5. Similarly, mark the upper and lower limits of the
blind spot.
6. You can map out the entire perimeter of the blind spot
with considerable accuracy, as the diagram indicates.

Exercise 5: Mechanical Stimulation of the Retina

The eye has properties similar to those of a camera, in that the image formed on the retina
is inverted. Light falling on the retina on one side of the eye gives a visual response in the
opposite side of the visual field. Mechanical stimulation of the retina, by pressure on the
eyeball, also gives a visual response that is inverted.

Procedure

1. Turn your gaze to the left, and shut both eyes. Keep looking to the left.
2. With a fingertip, press gently on the right side of your right eyeball, at the corner
of the eye. Note the visual effect.
3. Slide your finger up and down, and note the direction of movement of the visual
response.
4. Turn your gaze to the right, and similarly press on the left side of your right
eyeball, at the corner of the eye. Again, note the visual effect. You should find
that the main visual response to stimulation is a bright circle or disc, on the
opposite side of the visual field from the site of stimulation. Stimulation of the
retina on the right side of the eye gives a response on the left, and vice versa.

What did you observe when you mechanically stimulated your retina?

Exercise 6: The Positive After-image

Retinal photoreceptors have a surprisingly long and slow response to light. A brief visual
stimulus gives rise to a response that outlasts the stimulus long enough to give an after-
image.

Procedure

1. Face a bright scene, such as a sunlit window or a strongly illuminated bench-top.

2. Close both eyes and cover them with your hands. Wait for 30 seconds.
3. Remove your hands, and open your eyes for the shortest possible time, then close
them again.
4. Note the after-image. Bright features of the scene remain visible for an
appreciable time (a substantial fraction of a second).

Exercise 7: The Negative After-Image

The sensitivity of retinal photoreceptors decreases gradually while they are being
stimulated by light, and increases while they are not. This adaptation to light and dark
allows visual function over a very wide range of light intensities. It has the side effect of
giving rise to negative after-images.

Procedure

1. Place a black object on a piece of white paper, or draw a black square on the
paper.
2. Look fixedly at the black object for 30 seconds.
You may blink, but should take care to keep your
gaze fixed.
3. Shift your gaze to a piece of plain white paper, and
note the after-image of the black object. The image
lasts for many seconds. The image is inverted in
contrast (the black object gives a bright after-
image), hence the name 'negative after-image'.
4. Repeat with a colored object, and note the color
change in the after-image. For example, a red object gives a green after-image.

Exercise 8: Retinal Blood Vessels

The blood vessels of the retina lie in front of the neural and photosensitive layers.
Absorption and scattering of light by the retinal vessels would be expected to give rise to
an image. This image, however, is normally suppressed by poorly understood
mechanisms. Altering the direction of illumination of the vessels temporarily overcomes
this suppression, and makes the image visible. Sufficient light can enter the eye through
the sclera (the "white" of the eye) if a small, bright light source is held close to the eye in
such a way that little light enters via the pupil.

Procedure

1. Shut both eyes and direct the gaze to the left. Shine the beam of a small flash-light
on the eyelid at the right side of the right eye. Hold the flash-light close to the eye,
so that it forms a small bright illuminated spot.
2. A lacy network pattern should be visible for a short time, although it then fades.
Moving the flash-light slightly restores the pattern. Continuous rhythmic
movement of the flash-light keeps the image visible.
3. The vessels may be seen more clearly if the eyes are open and the flash-light is
directed at the sclera directly. This requires, however, either that the experiment is
done in a darkened room, or that the subject looks at a featureless scene, such as a
plain dark-colored wall.
 4. With patience, the branching zigzag pattern of the
vessels can be clearly seen. The center of the
visual field has no blood vessels passing across it.
Vessels approach, in a radial direction, from outer
parts of the field.
5. Most people can see the origin of the branching
pattern of vessels, in a small region to the right of
center. This is the optic disk (see Exercise 4).

Exercise 9: Visual Information

How much visual information does the brain need?

Procedure

1. Look at the image to the right. Can you recognise this

person?
2. Try screwing up your eyes (squinting). If you are short-
sighted, remove your glasses. Does this help?

Who did you see?

Exercise 10 : Optical Illusions

Several well-known optical illusions are shown in this section.

1. The Hermann Illusion.

While reading a book on sound by John Tyndall, L.

Hermann (1870) saw this illusion among the figures
that Tyndall had arranged in a matrix.

Observe Figure 1 to the right and describe what you

see.

What most people see:

Gray spots in the intersections of the spaces between

the squares. Every space in the grid has the same white
intensity, and yet the intersections appear gray.
How can this illusion be explained?

It has been argued that this illusion can be explained by reference to retinal receptive
fields. However, very recent research indicates that the explanation lies in cortical
interpretation of the information, not retinal signalling.

2. Size Perception

Observe Figure 2 to the right and describe what you see.

What most people see:

It appears that the blue line is definitely longer than the red line, but they are in fact of
equal lengths.

How can this illusion be explained?

The perceived size of objects is affected by a number of influences, of which the most
important is the angle subtended by the object at the eye. However many other cues are
used by the visual system, for example to decide if an object is small and near or large
and distant. The cues given by the black line ends in the image above confuse our
judgement of the line length.

3. An impossible geometric object

Observe Figure 3 to the right and describe what you see.

Figure 3. An impossible geometric object

What most people see:

Although at first glance this image 'makes sense' it quickly becomes obvious that it is an
impossible structure.

How can this illusion be explained?

The shadowing cues used here lure the brain into attempting to interpret this as a real
structure, but no such physical structure can exist.
Exercise 11: Colorblindness

As outlined in the Background section, about 5% of the male population has various
deficits in their ability to appreciate colors. The images presented here and on the next
page allow you to explore some aspects of this condition.

It is essential that you appreciate that the colors in the illustrations used here are not
reproduced with sufficient fidelity for them to be used for the clinical detection or
analysis of colorblindness. Should any of you think that you may have this problem based
on the illustrations here, you must see a specialist for a proper assessment.

1. Ishihara color vision test images.

The Ishihara color test is a test for red-green color deficiencies, the most common kind. It
was named after its designer, Dr. Shinobu Ishihara (1879-1963), a professor at the
University of Tokyo, who first published his tests in 1917. The full test consists of thirty-
eight plates. Nine images are shown here.

Procedure

1. Examine each image using the navigation buttons in the LabTutor.

2. Allow approximately 3 seconds to reach a decision about the number that you see.
3. Then click on the image to open another popup that will tell you what normal and
red-green color blind people would see.

Ishihara test image 1

Ishihara test image 2

Ishihara test image 3

Ishihara test image 4

Ishihara test image 5

Ishihara test image 6

 Ishihara test image 7

Ishihara test image 8

Ishihara test image 9

2. Examples of what colorblind people see.

There are mulitple types of colorblindness. The images to the left show what is seen in
some of these condition.

Procedure

Click through and examine each of the four images to the left.

Describe the color deficiency seen in each type of color blindness presented.
What a person with normal color vision would see

What a deutan would see

What a protan would see

What a tritan would see

Exercise 12: Two-Point Discrimination

In this exercise you will show that density of tactile receptors in the skin differs greatly in
different parts of the body.

Procedure

1. Take a metal paperclip and unfold it. Bend it into a U shape, with the wire points
about 10 mm apart.
2. Touch the two points gently on the palm of a volunteer's outstretched hand, and
ask if one point or two is felt. With a separation of 10 mm, the double stimulus
from the two points can be easily felt.
3. Ask the volunteer to close both eyes. Bend the paperclip so as to bring the points
closer together. By repeated trials with different point separations, find the
smallest separation that the volunteer can distinguish as two points. You can test
the truthfulness of the volunteer's responses, from time to time, by turning the
paperclip slightly, and pressing only one of the points down.
4. Measure the separation of the points with a ruler.
5. Repeat steps 3 and 4 with trials on different parts of the body (for example, a
finger tip, the back of the hand, the back of the forearm and the back of the body).
Complete the table on the next page.
 Two Point Discrimination

Location Minimum Distance to Feel two point

Palm

Back of Hand

Finger Tip

Back of Forearm

Back

Exercise 13: A Tactile Illusion

In this exercise you will demonstrate a tactile illusion.

Procedure

1. Cross two adjacent fingers over, so that the fingernails lie side by side, but in a
position reversed from the normal. Most people find it easiest to cross the middle
finger over the index finger.
2. Place a small object such as a pen in the V-shaped gap between the two
fingernails, and gently move it back and forth.
Exercise 14: A Thermal Illusion

Many sensory systems show adaptation: a declining response to a continued steady

stimulus. Temperature sensors in the skin adapt in this way, and so thermal sensations of
warmth or cold are determined more by changes in temperature than by the temperature
itself.

Procedure

1. Obtain three containers (small buckets or large beakers). Fill one container with
hot, but not painfully hot, water. Fill another with cold water, and fill the third
with lukewarm water.
2. Place one hand in hot water and the other hand in cold water. Leave them there
for 30 seconds.
3. Now place both hands in the lukewarm water.
4. Obtain a metal pipe and a wooden rod of similar length and thickness.
5. Place each in the cold water for about a minute, then remove them. Which feels
colder? Why?

Exercise 15: Taste and Smell

Both taste and smell use chemoreceptors. A large component of taste is actually due to
olfaction (smell).

Procedure

1. Ask a volunteer to close his or her eyes and to pinch the nostrils together,
preventing airflow through the nose.
2. Place a small previously prepared piece of apple in the volunteer's mouth, and ask
him or her to try to identify it by taste.
3. Repeat with a piece of raw potato, and then with a piece of raw onion.
Identification is extremely difficult.
4. Repeat steps two and three, but this time allow the volunteer to breathe through
the nose. Identification is now easy.
Exercise 16: Distribution of Taste Buds

Taste receptors ("taste buds") are found principally on the tongue, but also on the palate
and pharynx. Five kinds of taste bud are recognized: sweet, sour, salt, bitter and the
"umami" receptor that is sensitive to the amino acid glutamate . Each kind of taste bud
has a characteristic spatial distribution on the tongue.

Procedure

1. Prepare or get small containers of the following:

o Sucrose (table sugar) solution: approximately 15 g in 50 mL water.
o Sodium chloride (table salt) solution: approximately 5 g in 50 mL water.
o Citric acid: approximately 2 g in 50 mL water.
o Tonic Water: containing quinine, eg; Canada Dry or Schweppes Tonic
Water).
2. Dip a small piece of clean cotton wool or the end of a cotton bud in the sucrose
solution, then shake off the excess solution.
3. Apply the cotton to the back of a volunteer's tongue, and ask the volunteer to
report the sensation. Discard the cotton wool or bud.
4. Using a fresh piece of cotton wool or new cotton bud, test the sensitivity of one
side of the tongue. Similarly, test the tip of the tongue. The figure shows the
approximate regions that you should examine.
5. Repeat step two, but with the salt solution. Note the distribution of salt sensitivity.
6. Repeat step two, but with the citric acid solution. Note the distribution of sour
sensitivity.
7. Repeat step two, but with the 'bitter' solution. Note the distribution of bitter
sensitivity.

Describe your observations here.

Exercise 17: The "Joint Position" Sense

The capsule and ligaments of a joint receive a sensory innervation that is able to detect
changes in joint position. The effectiveness of this little-known sense is easily
demonstrated.

Procedure

1. Ask a volunteer to hold out one hand with the palm facing up and the fingers
stretched out.
2. Hold the volunteer's index finger by placing your thumb on one side and your
index finger on the other. Don't hold the volunteer's finger by the front and back;
that could give cues about movements, deriving from the force of lifting or
pulling down.
3. Bend the volunteer's finger up, while saying "This is up". Then pull the finger
down to the original extended position, while saying "This is down".
4. With the volunteer's eyes shut, test his or her ability to identify the direction of a
series of finger movements. Try both large and small movements.
Exercise 18: The Semicircular Canals

The semicircular canals are liquid-filled channels in the temporal bone of the skull, and
form part of the inner ear. They detect rotary movements of the head in three axes. This
exercise requires a swivel chair or a stool that can be rotated smoothly on its vertical axis.

Procedure

1. The volunteer should sit on the swivel chair, with both feet in the air, and close
both eyes.
2. Ask the volunteer to say when a rotation is detected, and to indicate in which
direction. Test the volunteer's ability to sense rotary motion, by rotating the chair
at various speeds and for various durations. You should find that even very slight
movements are reliably detected.

The semicircular canals detect rotation but do not signal the body's position. You can test
this by showing that the volunteer's idea of which direction he or she is facing becomes
unreliable after a sequence of rotations, such as a quarter-turn in one direction and then a
half-turn in the other. However, the test is not straightforward to do, because the
volunteer will make use of other directional cues. The ambient light intensity may change
with rotation, and this can be sensed even through closed eyelids. Acoustic cues are also
likely to be present in many laboratories.
 EXERCISE XIV

BLOOD

Objectives

1. To perform the following blood examinations:

a. Erythrocyte Sedimentation Rate

b. Hematocrit

c. Bleeding Time

d. Clotting time

e. Blood Typing

2. To compare the results of blood tests with the normal values

3. To discuss the significance of these blood examinations

Apparatus and materials

Wintrobe tube, test tubes, beakers, pricker, centrifuge machine, filter paper, NSS,
distilled water, anti-sera, 0.5% NaCl

Procedures

1. Erythrocyte Sedimentation Rate (Wintrobe and Landsberg method)

Extract 5 mL of venous blood from a volunteer and place in a tube with

anticoagulant. Then mix well by inversion. With a capillary hematocrit pipette, fill a
Wintrobe tube with blood to the “0” mark. This is done by passing a hematocrit pipette to
the bottom of the tube and gradually raising the pipette as it fills with blood. This helps
avoid the accumulation of air bubbles in the tube. The filled tube is then placed in an
exactly vertical position in an appropriate rack and left at room temperature for 1 hour.
The point on the millimeter scale to which the erythrocytes have settled is the ESR.
2. Hematocrit (Packed Red Cell Volume)

Perform a fingerstick and collect blood sample. Blood must be mixed with oxalate
thoroughly by not less than 30 slow and complete inversions of the container. After
adequate mixing, the hematocrit tube is filled using the filling pipette or syringe. Seal one
end of the hematocrit tube. To do this, place a finger over one end of the tube and gently
press the opposite end of the tube into the clay. Place the sealed tube in a groove of the
head of the microhematocrit centrifuge with the sealed end toward the outside of the head
and in contact with the rubber gasket. Balance the centrifuge by placing another tube in
the groove opposite the first. Cover down and lock the centrifuge. Set for 5 minutes.
After centrifugation, read the level of packed red cells without disturbing the specimen.
The result is calculated from the formula

Hematocrit (%) = L2 x 100

L1

3. Bleeding Time

After cleansing the volunteer’s fingertips with alcohol, allow blood to come out
by pricking the fingertip with a sterile lancet. Note the time of the first appearance of
blood. Wipe the bleeding area with filter paper. Do the wiping of the bleeding finger
from time to time until no more blood clings to the filter paper. Don’t squeeze the finger.
Using a timer, note the duration of time from the first appearance of blood until no more
blood is seen in the filter paper. This is the bleeding time.

4. Clotting time

After pricking a volunteer’s fingertips previously cleaned with alcohol and blood
comes out of the wound, start the timer. Place a drop of blood on a slide. From time to
time touch the drop of blood with the tip of the needle, noting the first appearance of
fibrin. Note the time when fibrin clings to the tip of the needle. That is the clotting time.

5. Blood Typing

A drop of blood from a volunteer is diluted with saline 10x so that clotting will
not occur. This leaves essentially a suspension of red cells in saline. Two drops of this
suspension is placed on a slide separately. A drop of alpha agglutinin is mixed with the
first drop and a drop of beta agglutinin is mixed with the second drop. After allowing
several minutes for agglutination to occur, determine whether the cells clumped by
observing under a microscope. If no clumping is noted on both drops, then the blood is
type O. Clumping in the first drop means the blood is type A. Clumping in the second
drop only means the blood is type B. Clumping in both means the blood is type AB.
Analysis

1. Give the results of the following blood examinations and compare with the normal
values.

a. ESR

b. Hematocrit

c. Bleeding Time

d. Clotting Time

e. Blood Typing

Study Questions

1. Define the following terms:

a. Erythrocyte Sedimentation Rate

b. Hematocrit

c. Bleeding Time

d. Clotting Time
2. Enumerate conditions or factors that may increase ESR.

3. Give the blood groups of the O-A-B system with their frequency or prevalence in the
general population.

4. Enumerate factors or conditions which may increase and decrease the hematocrit.