Skeletal muscle contraction: Lab Manual

Part I: Electromyography
Part II: Glycerinated muscle contraction

Learning Outcomes:

Students will:

• Gain an understanding of skeletal muscle structure and function

• Record EMGs while examining motor unit recruitment and fatigue
o Optional: listen to EMG “sounds” and correlate sound intensity with motor unit
recruitment
• Observe and measure contraction of glycerinated muscle under various
conditions
• Practice data collection, summary, and analysis
• Develop light microscopy (stereo dissection microscopes) skills
• Develop cooperative group skills and scientific writing skills

Preparation:

Moyes CD, Schulte PM. 2007. Principles of animal physiology. 2nd Edition. Toronto:
Pearson Benjamin Cummings. Chapter 5, Cellular movement and muscles; p. 208 –
235.
Pechenik JA. 2012. A short guide to writing about biology. 8th Edition. Toronto: Pearson.
Chapter 5, Citing sources and listing references; p. 69 – 79, Chapter 9, Writing
laboratory and other research reports; p. 172 – 182, p. 203 – 211.
• Carefully read the materials in this file.

Note: you will require lab coat, goggles, and closed toed shoes for this
laboratory. If you want to save your data and analyze it at home, please
bring a USB stick. You may also have the opportunity to email your file to
yourself.

Laboratory Outline:

The laboratory consists of two parts. Each laboratory section will be divided into two
groups. One group will begin the experiment with part I and the other will begin the
laboratory with part II. Half way through the laboratory, you will switch and complete
the other part of the laboratory.

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After the laboratory you must:

1. Individually complete the laboratory 2 assignment posted on Blackboard and

submit online by the deadline. The assignment includes:

a. Write an Introduction section (taking into account your TAs feedback on your
Pre-lab 2 submission).
a. Appropriate Title for a manuscript in which your introduction section
would appear. Refer to Appendix 3 of assignment for guidelines.
b. Your introduction should include rationale for conducting this
experiment, relevant background information (cited correctly: see
Appendix 2 of assignment for guidelines), brief explanation of how you
conducted the experiment, and your hypothesis.
c. You should include only three cited (referenced) sources: Pre-
laboratory 2 manual (this file), chapter 5 of Moyes and Schulte,
Principles of Animal Physiology, 2nd Edition, and one relevant peer-
reviewed journal article (chosen by you).
d. For assistance with your citations, please refer to Appendix 2 of the
Pre-lab assignment and Pechenik, p. 69-79.
e. To receive full marks, students must address the format and style
guidelines in Appendix 1 of the Pre-lab Assignment and in Pechenik
(pp. 203-211).

b. Answer a question related to your EMG experiment.

c. Create a figure illustrating your EMG experimental results comparing
dominant and non-dominant arm.

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Part I: Motor unit recruitment and fatigue.

Objectives:

• To determine the maximum clench strength for dominant and non-dominant

hands and correlate to differences in size of muscle.
• To observe, record, and correlate motor unit recruitment with increased force of
skeletal muscle contraction.
• To record the effects of fatigue on the EMG.

The measurement of the electric potentials generated by muscles is called

electromyography, and recordings of these potentials is called an electromyogram, or
EMG. The action potentials generated by contracting muscle alter the electrical charge
in the surrounding extracellular fluid. These electrical changes are conducted through
body fluids, and can be detected from the surface of the skin using electrodes applied
to the skin. The EMG signal is the recorded consequence of two principal bioelectric
activities: 1) propagation of motor nerve impulses and their transmission at the
neuromuscular junctions of a motor unit, and 2) propagation of muscle impulses by the
sarcolemma and the T-tubular systems resulting in excitation-contraction coupling. The
EMG is not a series of predictable waves like those of the electrocardiogram (ECG), but
a burst of spike-like signals. Integrating this complex signal will give an indication of the
intensity of muscle activity during a contraction. Although the electrical impulse
generated and conducted by each muscle fibre is very weak (less than 100 µvolts:
which is close to levels of background noise), many fibers in a motor unit conducting
simultaneously and multiple motor units induce voltage differences which are large
enough to be detected by a pair of surface electrodes and visualized after amplification.
Note that each fibre is a different distance from the electrode, and therefore signals
take different times to reach the electrode. Therefore, the surface EMG actually
provides indirect information about levels of motor unit activity. Furthermore, at any
instant in time the electrical potential between different parts of the muscle can be
either positive or negative, depending on which end of the muscle fiber is experiencing
the action potential. Thus, the voltage recorded can be either positive or negative, and
changes rapidly. EMGs can be used diagnostically to detect damage to muscle or to the
neural pathways responsible for triggering muscle contractions.

While you are collecting your EMG data, be sure to observe an interesting phenomenon,
which is hardly noticeable except when performing electromyography. That is tonus
(also called muscle tone), a constant state of slight excitation of a muscle while it is in
the relaxed, neutral state while avoiding fatigue. Tonus is due to alternate periodic
activation of a small number of motor units within the muscle by motor centers in the
brain and spinal cord. In skeletal muscles this aids in the maintenance of posture and in
the return of blood to the heart and serves to maintain the muscle in a state of
preparedness for response to a stimulus.

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In this section of the lab, you will examine motor unit recruitment and fatigue by
combining electromyography with dynamometry. Dynamometry refers to the
measurement of power (dyno = power, meter = measure), and the graphic record
derived from the use of a dynamometer is called a dynagram. You will correlate the
EMG signal with the power output of the muscle determined by a hand dynamometer
equipped with an electronic transducer for recording.

The Biopac system will display three windows of information:

1. The force you exert on the transducer

2. The electromyogram during contraction
3. The integrated EMG, which provides an indication of the activity levels of the
muscle.

Caution: If any discomfort is felt during the exercises, discontinue the experiment and
consult the instructor.

Note: if you would like to save a copy of your results, please bring a USB stick with you
to lab. You can download the student analysis software from Blackboard and analyze
your data on your own computer.

Note: to reduce the recording of background noise, it is important for the subject to
remain still (except for the clenching of the dynamometer). If the subject rests his or
her forearm on the table during the recording, less noise will be recorded from other
muscles in the body.

Setup and Preparation:

1. Check that your Biopac hardware is connected with the electrode set in Channel 1
and the SS25LA Dynamometer in Channel 2 (figure 1). You will also see
headphones connected to the rear of the Biopac hardware.

Figure 1. Biopac setup.

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2. Logon to the computer (choose student icon).

3. Turn on the MP36 (on/off button will be at the back).

4. Start the Biopac Student Lab 4.0 software.

a. Locate icon on the desktop and double click

b. The following screen will appear.

Figure 2. Biopac Lessons start-up screen.

c. Choose ‘Record or Analyze a BIOPAC Lesson’

d. Choose ‘LO2 – Electromyography (EMG) II’ and Click OK.

4. You will be prompted to choose, or name, a folder into which BSL will save your
lesson data (use a unique name such as ‘last name – Lab 2 – P0201C’. You will later
save your data to a folder on the desktop or onto your USB stick.

5. You can follow along to the protocol below. To further guide you, the LO2 lesson
has on-screen prompts and a lesson-specific HTML Journal with detailed
instructions for Setup, Calibration, and Recording, including embedded instructional
photos and videos.

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6. Choose one student to be the subject. The other student will help with instructions
and will complete the experiment after the first student.

7. Remove any jewelry or watches from your wrist. For the first series of exercises in
this lab, record from the subject's dominant arm.

8. Place electrodes on the skin of the subject’s dominant and non-dominant forearm as
shown in Figure 3. The electrodes are disposable adhesive disks. Peel off the
protective paper and stick them firmly in place. Do not remove the gel from the
surface of the electrodes–this is required to provide a good electrical connection.
Once the electrodes are in place do not readjust them because they readily lose
their stickiness.

9. Now clip the appropriately coloured electrical lead wires to the electrodes (refer to
figure 3). The pinch connectors work like a small clothespin and will only latch onto
the nipple of the electrode from one side of the connector.

White Lead (-)

Red Lead (+)

Black Lead (ground)

Figure 3. Location of EMG electrodes and leads on the forearm.

10. Subject should get into a seated comfortable position facing the screen. To
correctly grip the dynamometer, place the short grip bar against the palm, toward
the thumb, and wrap your fingers to center the force (see figure 4).

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 Figure 4. Grip position on dynamometer. IMPORTANT: Hold the dynamometer in the same position
for all measurements from each arm. Note your hand position and try to repeat it for all of your
experiments.

Calibration: The Calibration procedure establishes the hardware’s internal parameters

(such as gain, offset, and scaling) and is critical for optimal performance.

11. Click Calibrate and follow the prompts.

12. Verify that your recording resembles the example data.

• If similar, click continue and proceed to data recording

• If not, click Redo calibration
o If the data is noisy or flatline, check all connections to the MP unit.
o If the hand dynamometer signal is not zero when relaxed, make sure all grip force is
removed until prompted.
o Verify electrodes are making good contact and that leads are clipped to the correct color
position with minimal cable strain.

13. When Continue is clicked following Calibration, the display will change to show
only the Clench Force channel, with grids displayed.

You are now ready to collect data.

Four data recordings will be acquired, two on each arm:

a. Exercises 1 and 3 record Motor unit recruitment.

b. Exercises 2 and 4 record Fatigue.

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Protocol:

Exercise 1: Motor unit recruitment in Dominant Arm

1. Please completely read the following directions and/or review onscreen Tasks
menu before proceeding.

2. The subject should sit quietly with his or her dominant forearm resting on the lab
bench with an easy view of the computer screen.

a. Click Record.

3. Perform a series of Clench-Release-Wait cycles until maximum grip force is reached.

• Hold clench for three seconds, release for three seconds.

o It is important to reach the first gridline on the first clench. Increase grip on subsequent
clenches to advance the force signal one gridline per clench until maximum grip force is
reached.
o A total of five clenches are used in the Example Data, but certain Subjects may require
a lesser or greater number of clenches to attain maximum grip force.
o Completely relax grip force between clenches.

4. After maximum grip force is reached, click Suspend.

5. Verify recording resembles the example data shown.

• If similar, click Continue.
• If not, click Redo.

Exercise 2: Fatigue in Dominant Arm

Note the maximum clench force attained in Exercise 1 so you can determine when the
force has decreased by 50%. (The maximum force may scroll out of view.) Try to
maintain the maximum clench force. (The forearm will fatigue and the force will
decrease.)

1. Please completely read the following directions and/or review onscreen Tasks
menu before proceeding.

2. The subject should sit quietly with his or her dominant forearm resting on the lab
bench with an easy view of the computer screen.

b. Click Record.

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Note: the time to fatigue to 50% of maximal clench force will vary greatly among
individuals.

3. Clench the hand dynamometer as hard as possible and try to maintain maximum
force. Continue clenching until force has decreased by 50%.

4. Click Suspend.

5. Verify recording resembles the example data shown.

• If similar, click Continue.
• If not, click Redo.

Exercise 3 and 4: Motor unit recruitment and Fatigue in non-Dominant Arm

1. Refer to figure 3 for correct placement of electrodes on the non-dominant forearm

and for the correct connections of the colour-coded leads.

2. Prepare to repeat exercises 1 and 2 for the non-dominant forearm.

3. Verify recording resembles the example data shown.

• If similar, click Stop.
• If not, click Redo.

Optional: Listen to EMG and correlate intensity of sound to intensity of clench.

1. Place headphones over your ears and click Listen. Grip and depress the hand
dynamometer.
2. Click Stop to end the optional EMG listening exercise.
3. Click Done to end the lesson.

4. You will now see the screen shown in figure 5.

a. To record from another subject choose ‘Record from another subject’ and click OK.
b. To analyze current data choose ‘Analyze current data file’ and click OK.
c. To copy your file to another location choose ‘Copy to another location’ and click
OK. Save the file type as *.acq. Choose a unique file name such as ‘lastname-pre-
lab2-P0101A’ and save it in your lab folder File > Save as > Desktop >
BIO270Lab2 EMG Data > choose your lab section (i.e. P0101B), or on your USB
stick File > Save as > your USB.

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 Figure 5. End of Lesson choices.

Data Analysis:

Refer to the Analysis Tool guidelines provided in the lab for a reminder of the tools
available to you. You may complete your analysis in the lab or choose to take your data
home to analyze on the Biopac analysis software downloaded from blackboard. If you
choose this option, ensure that the program works on your computer.

To analyze previously saved data. Open the Biopac software and choose ‘Review Saved
Data’ (figure 2). Locate your file and begin.

Note Channel Number (CH) designations (these are slightly different from the prelab):

CH 1 EMG (Hidden)
CH 40 Integrated EMG
CH 41 Clench Force

1. Setup your display for optimal viewing of “Dominant arm: Increasing clench force”
data.

2. Read the journal and note your force increment in the Data Report.

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3. Use the I-Beam cursor to select an area on the plateau phase of the first clench
(Figure 6). Record your data in the Journal. You may use the existing tables if you
choose. Right click in any box in the tables and follow the instructions. You may
also copy and paste your data into an excel file.

4. Repeat Step 3 on the plateau of each successive clench.

Figure 6. Selecting plateau of clench.

5. Repeat these steps for the “Non-dominant arm: Increasing clench force” data.

6. Scroll to “Dominant arm: Fatigue data” and set up your display for optimal viewing.

7. Use the I-Beam cursor to select a point of maximal clench force immediately
following the start of the recording (Figure 7).

Figure 7. Selecting maximal clench force.

8. Calculate 50% of the maximum clench force from Step 7.

9. Find the point of 50% maximum clench force by using the I-beam cursor and leave
the cursor at this point.

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10. Select the area from the point of 50% clench force back to the point of maximal
clench force by using the I-beam cursor and dragging (Fig. 2.19). Note the time to
fatigue measurement (CH 40 Delta T). Record your data in the Journal. You may
use the existing tables if you choose. Right click in any box in the tables and follow
the instructions. You may also copy and paste your data into an excel file.

11. Repeat these steps for “Non-dominant arm: Fatigue data”.

Optional:

12. Using a string and ruler, measure the circumference of your dominant and non-
dominant forearms at their greatest girth and record.

13. Compare the measured circumference of your dominant and non-dominant arms to
their maximum clench forces and to their times to fatigue. Is there a correlation? Is
there a correlation with others in the class?

14. If time permits, begin designing a figure illustrating motor unit recruitment in your
dominant and non-dominant arms (see Lab 2 Assignment, Q. 2). Be sure to
correctly label your axes and include a relevant figure title in the appropriate
position (refer to Pechenik pp. 164-195).

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Part II: Glycerinated Muscle Contraction

Introduction:

Fresh muscle fibres are difficult to experiment with because they deteriorate rapidly. As
a result, we will use muscle fibers that have been treated and stored in glycerol. Cell
membranes can be artificially removed using a variety of chemical solvents such as
glycerol. The glycerination process removes ions and ATP from the tissue. In addition,
the regulatory proteins troponin and tropomyosin are disrupted and no longer block the
binding of actin and myosin, so that calcium is not required to induce contraction.
However, since ATP is not present, the myosin heads are not energized.

The advantage of such preparations is that it allows for study of the intracellular
components, without disrupting the integrity of their structure and position in a cell,
and without having to stimulate intracellular signaling pathways. It allows scientists to
control the composition of the intracellular environment without destroying the
contractile machinery.

The purpose of this simple experiment is to witness and measure the contraction of
skeletal muscle (Rabbit psoas muscle), and to observe the effects of different
intracellular ions and ATP on the ability of actin and myosin to undergo power strokes
(contract). Rabbit psoas muscle is a strap muscle composed of mixed fibre type (fast
and slow twitch muscle fibres) that assists during rotation of the hip.

Note: It is believed that KCl solution is used to restore the normal ionic environment of
the muscle cell, since potassium is the major cation found in the intracellular fluid and
magnesium is an ion that is required for ATP to bind to the myosin head.

Solutions:

1. 5 mM ATP (approx. 0.25%) in ddH 2 O.

2. 100 mM KCl, 4 mM MgCl 2 in ddH 2 O.
3. 5 mM ATP, 100 mM KCl, 4 mM MgCl 2 ddH 2 O.

Note: Do not contaminate the glycerinated muscle with outside ions such as metal, Ca2+
or salts of any kind. Care must be taken to avoid any possibility of cross
contamination between the ATP and salt solution vials. Any contamination will lead
to ambiguous experimental results. Wash your hands before starting.

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Procedure:

1. Ensure that you have the following on your lab bench: a microscope, 3 slides, a
transparent ruler, 2 teasing needles, three labeled plastic pipettors, eppendorf
tubes of Solution 1, 2 and 3 (ATP and salts).

2. Retrieve a fascicle of muscle tissue in glycerine in a petri dish (do not let your
muscle dry out; keep it in glycerol).

3. Using the teasing needles, tease the muscle fragment you obtained into separate
fibres. The goal is to get single muscle fibres (i.e., muscle cells), although this may
be difficult. In any case, each strand must not be more than 0.2 mm in diameter.
Single fibres will demonstrate the greatest contraction response.

4. Place three single muscle fibres with a small amount of the glycerine on each of the
3 microscope slides. Transfer the minimum amount of glycerol possible and array
the strands so that they are separated, straight, and parallel to one another.

Note: The amount of glycerol needed depends on the heat of the microscope
lamp and the length of exposure to heat. The less glycerol used, the easier the
fibers are to measure and the greater the contraction. The glycerine simply
ensures that the muscle fibres do not dry out. A thin layer on the slide before
transfer will be sufficient.

5. Label your slides 1, 2, and 3.

6. Arrange the fibres in a parallel fashion on each slide.

7. Measure the length of each fibre by placing the slide on top of the ruler under the
microscope.

8. Add three drops of ATP solution to slide 1. Immediately observe the fibres under
the microscope.

Note: add these drops gently as you do not want to disrupt the location of your muscle
fibres.
9. After 2 minutes, re-measure and record the length of the fibres. Gently straighten
the fibre out using the needles, but do not pull or stretch the muscle fibre. Measure
and record the final muscle fiber length.

Note: do not cross contaminate the solutions. Use only the designated droppers.
Remember to rinse your dissecting needle with ddH 2 O between solutions to
minimize cross contamination.

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10. Add three drops of Salt solution (containing K+ and Mg2+) to slide 2. Immediately
observe the fibres under the microscope.

11. After 2 minutes, re-measure and record the length of the fibres. Gently straighten
the fibre out using the needles, but do not pull or stretch the muscle fibre. Measure
and record the final muscle fiber length.

12. Add three drops of ATP + Salt solution to slide 3. Immediately observe the fibres
under the microscope.

13. After 2 minutes, re-measure and record the length of the fibres. Gently straighten
the fibre out using the needles, but do not pull or stretch the muscle fibre. Measure
and record the final muscle fiber length.

14. Calculate the percent contraction for each of the nine fibres using the following
formula:

% change = ǀFinal length (mm) – Initial length (mm)ǀ

Initial length (mm) X 100

Note: indicate that your result was a reduction in fiber length by including a ‘-ve’
sign in front of the percent change.

15. Calculate the mean of the three fibres for each solution and add to the class data
table. Ensure that you save your data after you add to the table. This file will be
posted to Blackboard. You will need it to analyze your data and it will be used as
the results on which to base your Introduction. Ensure that you use your lab
section’s data.

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