1.

IMMUNOLOGY
● Purpose:
1. Demonstrate how the ELISA assay is used to diagnose exposure to a disease
a. By testing for the presence of antibodies to the disease in a sample of
simulated serum
2. Determine the hemagglutinating antibody titer of two different sera
a. From two different patients
3. Demonstrate complement mediated lysis
a. And the sequence of reactions necessary to cause lysis
● Basic Concepts:
○ ANTIGEN
■ A toxin or other foreign substance
■ (non-self) cells and molecules
○ When a foreign antigen is introduced into an animal, the animal will respond
immunologically to it
1. Innate (natural) responses
a. Occur as many times the foreign agent is encountered
b. LACKS immunological memory
2. Acquired (adaptive) responses
a. Improves based on repeated exposure to the antigen
■ Innate and acquired responses usually work together
○ The immunological response may be or TWO different types:
1. Cell-mediated or
2. Humoral (relating to body fluids)
● Immune System
○ NOT localized in one location
■ Very large and diffuse system
○ Linked by the circulatory system and lymphatic vessels
■ What brings the different parts of the body together
● Allows cells to move from place to place
○ Lymph
■ A colorless fluid containing white blood cells
■ Bathes the tissues and drains through the lymphatic system into the
bloodstream

○ Organization

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■ The cells involved in the immune response are effectively organized
into tissues and organs
■ Major lymphoid organs are classified in two categories:
● PRIMARY
○ Major sites of lymphocyte development → lymphopoiesis
■ Lymphocyte
● A form of small leukocyte (white blood
cell) with a single round nucleus
○ Where cells are BORN and DEVELOPED
○ Function
a. To produce a LARGE repertoire of cells
i. B & T-cells
ii. Able to recognize and respond to a variety
of ANTIGENS
iii. Acquire their individual repertoire of
specific antigen receptors
b. To ELIMINATE any self-reactive cells
i. Don’t want cells of the immune system to
attack the tissues of the body itself
ii. Cells with receptors for autoantigens are
mostly eliminated
1. Autoantigens → “self” antigens
1. Thymus
a. T cells will differentiate here
2. Bone Marrow
a. B cells will differentiate here
b. Where all the blood cells are produced
i. Where they start dividing
● SECONDARY
1. Spleen
2. Lymph Nodes
3. Mucosal Associated Lymphoid Tissue (MALT)
4. Bronchial Associated Lymphoid Tissue (BALT)
5. Tonsils
6. Adenoids
7. Peyer’s Patches
8. Gut Associated Lymphoid Tissue (GALT)
■ Function
● Provide an environment for:
i. Proliferation of immune cells
1. Rapid increase in numbers
ii. Maturation of immune cells
● Filter and trap different antigens
● As they go through the circulatory system
● Provide an environment for:
○ Cell-Cell interaction
○ Between the different cells of the
immune system

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 ○ Cytokine-Cell interaction
■ Cytokines
■ Excitatory chemicals
released by some of the
activated immune cells
■ Have an effect on other cells
● CELLS of the Immune System

○ Immune responses are mediated by a number of cells and by the soluble

molecules they secrete
○ Origin
■ All the cells shown above arise from the hematopoietic stem cell
■ Eg.
1. Platelets
a. Released into the circulation
2. Granulocytes and Monocytes
a. Pass from the circulation into the tissues
b. Mast cells are found in all tissues
i. Inflammatory mediator
3. B cells
a. Mature in the blood marrow
4. T cells
a. Mature in the thymus
5. Lymphocytes
a. Leave primary lymphoid organs and recirculate through
secondary lymphoid tissues
6. Interdigitating and Dendritic cells
a. Act as antigen-presenting cells in secondary lymphoid
tissues
○ Overall Function of these Cells
1. The innate/natural responses use phagocytic cells
a. Eg.
i. Neutrophils
ii. Monocytes
iii. Macrophages
b. These release inflammatory mediators and natural killer cells
i. Inflammatory mediators:

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 1. Basophils
2. Mast cells
3. Eosinophils
c. The molecular components comprise complement, acute-phase
proteins AND cytokines (eg. interferons)
2. Acquired (adaptive) responses involve the proliferation of
antigen-specific B and T cells
a. Occur when their surface receptors bind to the antigen
b. Specialized cells (antigen-presenting cells) display the antigen to
T lymphocytes and collaborate with them in the response to the
antigen
■ Major Histocompatibility Complex (MHC)
● A set of genes that code for cell surface proteins
● Essential for the acquired (adaptive) immune system to
recognize foreign molecules in vertebrates
■ This, in turn, determines Histocompatibility
● Compatibility between the tissues of different individuals
■ Was first identified when it was observed that histocompatibility
DEPENDED on the donor and recipient sharing the same haplotype
● A set of DNA variations that tend to be inherited together
● The molecules which determine GRAFT REJECTION are a limited group:
1. Class I
2. Class II
● The molecules which present antigens to T-cells are mostly encoded
within the MHC
● ALL nucleated cells of the body express MHC Class I
○ By contrast, MHC Class II molecules are used by antigen
presenting cells to present antigens to helper T-cells
■ So, cells expressing Class II are smaller in numbers than
those expressing Class I
○ ANTIGEN Presenting Cells
■ Otherwise known as an “Accessory Cell”
● A cell that displays antigen complexed with major
histocompatibility complexes (MHCs) on their surfaces
○ This process is known as antigen presentation
■ Macrophages
● Mononuclear Phagocytes
○ Most important group of long-lived phagocytic cells
● These cells are bone marrow-derived
● Function
1. Engulf particles
2. Internalize them
3. Destroy them
■ Dendritic Cells
● Found in the T-cell areas of lymph nodes and spleen
● The MOST effective cells for the initial activation of naive T-cells
■ B-cells
● Bone marrow-derived

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 ● Recognize FOREIGN ANTIGENS
○ Secrete immunoglobulins (antigen specific antibodies)
■ Responsible for eliminating extracellular
microorganisms or foreign agents
● Each B cell is genetically programmed to encode a surface
receptor specific for a particular antigen
○ Vast repertoire, each with different receptors on their
surface
● Upon recognition of the antigen:
○ B cells multiply and differentiate or turn into plasma cells
● These produce large amounts of the receptor molecule in a
SOLUBLE form that can be secreted → ANTIBODY

■ Germinal Centre
● With the initiation of the acquired immune response, germinal
centres form in the secondary lymphoid tissues
● Where all the antigen-specific and antigen-presenting cells can
interact
○ Effector Cells
■ Relatively SHORT-LIVED activated cells that defend the body in an
immune response
■ T Cells
● There are several types of T cells with a variety of functions
● They originate from bone marrow stem cells
○ BUT, require further differentiation in the THYMUS
(where they migrate)
● T cell maturation requires a number of cell interactions:
○ T cells express (in an orderly fashion) certain markers or
cell surface proteins

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 ○ Cell surface molecules are characterized on the basis of
their reactivity to monoclonal antibodies
■ The nomenclature used to refer to cell surface
molecules follows the “cluster of differentiation"
numbering
■ CD4+ T cells → CYTOKINE-secreting helper cells
1. Type I helper T cells
2. TH1
a. Secrete Interleukin (IL) 2 and interferon
(IFN) g
3. The production of cytokines facilitates
cell-mediated immunity
a. Activation of macrophages and T-cell
mediated cytotoxicity (toxic to cells)
4. Type II helper T cells
5. TH2
a. Secrete IL4 and IL5
6. Help B cells produce antibodies
a. Class II MHC on antigen presenting cells
interact with CD4 on T cells
7. Peptides which bind to MHC Class II come
from proteins which have been internalized
by the cell and then degraded

● CD8+ T cells → play a role in the ELIMINATION of virally infected

cells
○ The infected cell marks itself as a target by displaying
peptides
■ Derived from intracellular viral protein on its
surface
○ Viral proteins are bound to peptide-binding regions of
class I MHC molecules
■ Peptides which bind to MHC Class I molecules
come from proteins synthesized within the cell
(which are broken down)
■ Transported to the endoplasmic reticulum
■ B Cells

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● The earliest cells which develop are called B1 cells
○ They express CD5 cell-surface molecule and are the
source of "natural antibodies"
■ Are IgM antibodies and are frequently polyreactive
● Recognize different antigens, pathogens
and autoantigens
■ Natural antibodies have a relatively low affinity
● Most B cells lack the CD5 molecule
■ They develop later and are called B2 cells
● Mature B2 cells
○ Coexpress IgM and IgD antibodies on their cell surface
■ The genes encoding B-cell receptors undergo a
process of somatic hypermutation
○ The final stages of differentiation of B2 cells into
antibody secreting plasma cells occurs
■ Within the germinal centres of secondary
lymphoid tissues
● To elicit a strong antibody response, B cells require:
1. Antigen
2. T cells
a. For direct contact
b. Usually TH2 cells
3. Soluble cytokines
a. E.g. IL4 + IL13, INF-γ or IL10
4. Certain adhesion molecules
● Clonal selection involves the proliferation of cells which
recognize a specific antigen
○ Each B cell is programmed to make just ONE antibody
specificity
■ Located on its surface as an antigen receptor
○ Antigen binds to only those B cells with the appropriate
surface receptor
■ These cells are stimulated to proliferate and
mature into antibody producing cells
○ When a B cell comes in contact with the antigen (foreign
particle) for which it has a receptor:
■ Leads to a cascade of activation of that specific
type of B-cell
■ That B-cell starts dividing
● Due to an interaction with a T-helper-cell
● Which releases cytokines that activate the
division of the B-Cell
○ When it divides, it produces a lot of plasma cells
■ Which are ANTIBODY FACTORIES
● Life span: 7-10 days
■ While they’re alive, they constantly pump out
antibodies

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 ● The antibodies are specific to the antigen
that they were activated by in the first
place
○ Also produces a lot of memory cells
■ Can respond much more quickly if the body
encounters the same pathogen again
● BASIS OF IMMUNIZATION
○ Very important!!!
■ Plasma cells produce antibodies → antibodies
attach to antigens → signals to phagocytic cells to
engulf them
○ Amplification of the response
■ Body is ready for action if the antigen is
encountered for a second time
● Basic mechanism:

● How do we know if someone has been exposed to a disease?

○ Measure blood to find how many antibodies are present in the blood for a
certain antigen
■ If there are none, the person has never encountered the disease
● Give a “booster shot” to increase their antibody titer
■ If there are some, the person has probably encountered it
● In labs, antibodies can be made that can recognize almost anything that we want
○ Eg. identify a specific ion channel
■ Can take tissue slices of the brain to see where the ion channels are
expressed
■ Put the antibodies on the brain slices
● Use a second antibody that has a fluorescent tag
■ Look under the microscope and see where the ion channels are located
● Definitions
1. Serum

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 a. Plasma minus the clotting factors
i. Fluid component of uncoagulated blood
b. Lots of antibodies or immunoglobulin
c. Individual has NOT been exposed or immunized with the antigen
2. Antiserum
a. Individual is exposed or immunized with the antigen
b. Serum that contains SPECIFIC antibodies to a specific antigen
3. Immunoglobulin
a. Similar to antibody
b. Attached to B-cell membrane (transmembrane domain)
c. No immunization
4. Antibody
a. An immunoglobulin produced in response to a SPECIFIC ANTIGEN
i. Can bind to that specific antigen
b. Making an antibody requires immunization
c. Function
i. Bind to the antigen
1. The antibody directs specificity towards the antigen
ii. By doing so, it also arms the killer cells and activates
complement:
1. Processes that eliminate foreign organisms
iii. The antibody cannot do this by itself
d. Structure

i. Two heavy chains (Y-shaped)

ii. Two light chains
iii. Constant region/Fc region
1. Defines what CLASS of antibody it is
a. “Handle” of the antibody
2. Immunoglobulin subtype
3. IgG
a. Most of the antibodies in our bodies are IgG’s
b. Major class of antibodies in our body
c. The most important class of immunoglobulin in
secondary immune responses
d. “Single units”

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4. IgD
a. N/A
b. “Single units”
5. IgE
a. Involved in allergies
b. “Single units”
6. IgA
a. Present in secretions
b. Eg. breast milk
i. Baby gets IgG from the mother through
the placenta
1. For the first six months of life,
they’re protected by antibodies
made from the mother
ii. After a while, those antibodies will be
diluted out and lost (lose their efficacy)
1. Baby’s system will now take over
and take care of the immune
responses
c. “Single units”, but can also be “dimers”
7. IgM
a. Pentamers
b. During the initial immune response, these are the
first things that appear (predominant antibody)
i. Found in primary immune responses
c. Also associated with the immune responses to
antigenically complex, blood-borne agents
d. Once bound to the antigen, it is a powerful
activator of the classical pathway complement
i. A single molecule of bound IgM is able to
initiate the cascade because of the
adjacent positioning of the Fc regions
e. Not as effective as immune fighting agents
i. Later, when the B-Cell becomes fully
activated, IgG molecules are released

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 8. The exposure to a foreign antigen yields a biphasic
response
a. First Phase
i. Associated with production of IgM
ii. Followed by production of IgG
b. Second Phase
i. Characterized by a reduction of IgM
followed by an increase of IgG
ii. The antigen will select and expand a clone
of effector B cells
1. Will develop into plasma cells and
produce antibodies
iv. Variable Region
1. What makes them respond to a SPECIFIC antigen
a. Great diversity from one cell to another
2. In terms of which specific receptors are present on its
surface AND which antibodies are produced
● Course of a Typical Antibody Response:

1. Lag Phase
a. Time where no response is seen
2. Primary
a. First response usually results in low levels of antibodies produced
i. Will eventually decrease
b. Produced 5-7 days after immunization
3. Secondary (subsequent exposures)
a. Faster rate and amount of antibodies produced
b. Response is SPECIFIC to the antigen
i. Just because the body can respond to antigen A faster, does
NOT mean that it will affect antigen B in any way….
■ Mechanisms (that underlie this phenomenon):
1. PREEXISTENCE of memory cells
a. Formed after the primary immune response

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 2. LARGER numbers of specific clones
a. These are ready to proliferate and differentiate on
demand
○ RATIONALE for immunization!!!!!
● How do antibodies MEDIATE their effects:
1. Neutralization
a. Antibody can bind to the toxin and neutralize it
i. Toxin is no longer effective
b. Shape has changed
i. Can no longer bind to something
c. Antibody binds to different pathogens (eg. bacteria)
i. Can no longer gain entry into cell
2. Macrophages (dendritic cells, neutrophils)
a. Engulf and digest antibody-bound pathogens
3. Complement Proteins/System
■ Family of PLASMA proteins
● Always present in our blood
■ Consists of about 20+ serum molecules
● Some of them are proteases
○ An enzyme that catalyzes proteolysis (the breakdown of
proteins into smaller polypeptides or single amino acids)
■ Constitutes nearly 10% of the total serum proteins
■ Forms one of the major defence systems of the body
● Antibodies can bind to these molecules or proteins present in
the blood (complement)
■ Functions:
1. Vasodilation
a. Increases the permeability of the capillaries
i. So lots of immune cells can enter the site of injury
b. Factors
i. Some of the protein fragments of the
complement system (C5a) causes degranulation
of mast cells and basophils
1. With release of histamine and other
vasoactive mediators
ii. Consequently, there are indirect effects on blood
vessels, vasodilation and increased permeability
of capillaries
2. Chemotaxis
a. Signals that attract immune cells to the site of injury
b. Factors
i. Polymorphs and macrophages have specific
receptors for small complement fragments
1. Generated during complement activation
ii. The fragments diffuse away from the site of
activation and stimulate chemotaxis
1. The same way chemokines do
3. Enhancement of Phagocytosis

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 a. By attaching complement molecules to the bacteria
b. Causes opsonization
i. Particles (eg. bacteria) are targeted for
destruction
ii. Makes it easier for the bacterial cells to be seen
by the phagocytes and eaten
1. Phagocytic cells carrying receptors for the
complement components are then able to
bind to the foreign particle
iii. This triggers phagocytosis and cell activation
4. Membrane Attack Complex (MAC)!!!!!
a. Produces direct destruction of cells
b. The final step in complement activation brings about the
assembly of a membrane attack complex
i. Can insert itself into lipid bilayers, causing lysis of
the foreign body
c. Process
i. Specific antigen and antibody binding occurs
ii. Ag-Ab complex:
1. Antibody-antigen complex produces a
cascade effect called a complement
cascade (can kill microbes)

■ Engages/activates C1 (complement 1)
● Causes a cascade of several other cascade
molecules
■ C5-C9 come together
● Puts a hole or pore in the
bacterial cell, which kills it
■ The hole allows the inside of the
bacteria to leak out and the
extracellular fluid to leak in
● LAB
1. HEMAGGLUTINATION
a. Agglutination (clumping) involving RBCs
b. Antibodies can be detected in the serum of animals
i. If RBCs are used as a source of antigen, the assay is called
hemagglutination

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c. In the experiment, SRBC are added to serially diluted serum from the
immunized animal
i. An animal receiving Sheep red blood cells (SRBC) will develop
antibodies against SRBC
ii. The antibodies in the serum will agglutinate to SRBC added in a
test tube
1. Results in a specific looking pattern at the bottom of the
tube
iii. Forms a MAT rather than a pellet
d. Originally used in blood banks
i. To ensure a blood recipient does not have preformed antibodies
to the blood cells of the donor
e. What are we doing?
i. Measure the concentration of antibodies in an antiserum relative
to a particle (RBC)
ii. Hemagglutination is expressed as the Titer
1. Concentration of antibodies where we get
hemagglutination
a. Inverse of the lowest dilution of the antibody in
the sample that is positive (+) for
hemagglutination (matting)
b. e.g: If hemagglutination occurred at dilution of
“1:8”, then the titer would be 8
2. Need to know:
a. Which wells are (+) and which are (-)
b. CONCENTRATION of the starting serum
c. The series of steps you are doing to make the
DILUTIONS
i. So we know what concentration of each
well is
iii. 2-fold serial dilution of our antibody sample
1. Method of diluting a stock solution where the
concentration decreases by the same quantity in each
successive step
iv. Start out with full strength, undiluted serum or antiserum
1. Take 1 mL of that, move it to another well (mix it up and
down)
2. Repeat
v. Each time, we reduce the concentration by half (2-fold)

vi. Doing this in 96-well plates

vii. Using pipetman (P200 and P1000)

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 1. Used to deliver small, specific volumes
2. Used for mixing
a. Best mixing by pushing and releasing the push
button gently and smoothly
3. P200 → volumes up to 200 ul (microliters)
4. P1000 → volumes up to 1000 ul
a. This is the maximum volume
i. If you turned it past 1000, the pipetman
would be damaged
b. Multiple by 10 (add another 0) to the number
displayed
i. Eg. 101 = 1010 ul
5. Do not push past first stop when taking up liquid
a. Will take up more solution than you wanted
f. Procedure:
i. Two sera (serums) are given
1. One from a "normal" mouse
2. One that was immunized with SRBC
ii. Using microtiter plates, place 0.1 ml of balanced salt solution
into each well of rows A and B
1. Add 0.1 ml of serum #1 to the first well
2. Mix and transfer 0.1 ml to the next well in row A and so
on until you reach the end
iii. Repeat the procedure for serum 2 in row B
iv. Add 0.1 ml of a 1% suspension of SRBC
v. Incubate for 30 minutes at 37C and then place in the
refrigerator
1. Best results are obtained if you examine thhhe plates the
next morning; however you should have results by the
end of the laboratory session
2. Explain your results and report the titer of the two
antisera
vi. Serial dilution (doubling dilution)
1. Dilute a reagent or compound in series or in sequence
a. Since you take the same quantity of reagent from
one well to the other, and the first well has the
same volume as you put in:
2. You are DOUBLING your dilutions

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g. Results? (what should we see)
i. Antibodies may be detected and measured by hemagglutination
at LOWER concentrations than those detectable by other
techniques
1. Relies on the ABILITY of the antibodies to cross-link red
blood cells by interacting with the antigens on their
surface
ii. The agglutination of an antigen (result of crosslinking with
antibodies) is dependent on the correct proportion of antigen to
antibody
iii. Reading the 96 well plate:
1. If sufficient antibody is present to agglutinate and form
cross-linking with the antigen:
a. Results in a visible mat formed by Ag-Ab complex
crosslinking
b. Zone of Equivalence
i. When the correct proportion of antibody
to antigen occurs
2. At TOO LOW of a concentration of antibodies:
a. RBCs roll down the sloping sides of the well to
form a red pellet or "button" at the bottom of the
well
3. At TOO HIGH of a concentration of antibodies:
a. The antibodies will completely surround the RBCs
and a mat will not form
b. Ag-Ab complex cross linking is prevented to occur
i. Every epitope on one antigen particle may
bind to a single antibody molecule

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 h. Questions to consider:
i. At what concentration do we get hemagglutination?
ii. At what concentration does it fall off?
1. When does the concentration of the antibody become
too low to cause hemagglutination?
iii. At higher dilution of serum, agglutination may occur:
crosslinking is possible. Why?
iv. Testing serum at only one concentration may give misleading
conclusions. What might the absence of agglutination reflect?
2. COMPLEMENT-MEDIATED LYSIS / CYTOTOXICITY
a. An effector function assay
i. Antibodies, by themselves, are a rather ineffectual in eliminating
foreign organisms
b. However, antibodies of the IgM and IgG class can activate the
Complement (C') system
i. Results in a stimulation of different effector functions
1. Eg. phagocytosis and lysis of foreign organisms
ii. Formation of the Membrane Attack Complex (MAC)
c. If red blood cells are used as a source of antigen, it is relatively simple
to demonstrate the lytic properties of the At-Ag complement complex
i. Not really used nowadays..
1. But previously, it was used in research to test for the
presence of an antibody to cell surface antigens
ii. Used for HLA typing
1. If you were going to have a rejection of a certain graft
d. Purpose
i. Find what are the conditions you need in order to produce a
membrane attack complex
ii. This experiment will demonstrate that you need an
antibody-antigen complex in order to activate SRBC lysis (sheep
RBCs)
e. Procedure
i. Take 5 tubes and label them 1 to 5
1. In each tube place 0.3 ml of SRBC (1% solution)
a. In tube 1 place 0.6 ml of saline
b. In tubes 2 and 3 place 0.3 ml of antiserum

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 c. In tubes 2 and 5 place 0.3 ml of saline
d. In tube 4 place 0.3 ml of normal mouse serum
2. In tubes 3, 4 and 5 add 0.3 ml of complement solution
a. Place in the water bath at 37C and incubate for
30 minutes
3. Remove from the water bath and centrifuge
4. Record the colour of the solution in each tube and
explain the reason for the differences observed

ii. After recording your results, take tubes 2 and 4 and add 3 ml of
saline to each tube and gently resuspend the RBC pellet with a
Pasteur pipette
1. Centrifuge them gently for 5 minutes to form a RBC
pellet
2. Remove the tubes from the centrifuge and aspirate off
the supernatant with a Pasteur pipette
iii. To each tube add 0.3 ml of saline
1. To tube 2 add 0.3 ml of C' solution
2. To tube 4 add 0.3 ml of antiserum
3. Mix gently with a Pasteur pipette and incubate at 37C
(water bath) for 30 minutes
a. Centrifuge for 5 minutes
4. Record the colour change and interpret your results

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 f. Results?
i. Upon centrifugation, the cells "made up" in clear saline will
collect into a pellet at the bottom of the tube
1. Since no lysis has occurred, there will be a clear saline
supernatant over the pellet (left tube)
ii. If lysis has occurred, the release of sheep red blood cell content
in the supernatant will occur
1. A "ghost" pellet made up of cell membrane debris will be
at the bottom of the tube (right)

g. Question to Consider
i. What do you conclude about the specificity of antibody and
complement binding?
ii. Which sequence of events caused cell lysis?
3. ELISA Antibody Test
a. Enzyme-Linked ImmunoSorbent Assay Antibody Test
i. A serological test
1. Study of serum and other bodily fluids
b. Used to detect particular antigens or antibodies
i. Antibodies in serum that recognize a specific antigen
ii. Exposure to a disease-causing pathogen
1. Eg. used to diagnose HIV infection by detecting the
presence of antibodies to HIV
c. Methods:
i. A few methods exist
1. Differences reside in the sequence in which antigens and
antibodies are added to a solid substrate
ii. AntiBODY capture assay
1. Antigen is first bound in the plastic wells
a. Then, the primary antibody binds to (or is
captured by) the immobilized antigen
b. A secondary antibody (conjugated to the enzyme
horseradish peroxidase (HRP)) is added, and binds
to the first antibody
2. The substrate 3,3’,5,5’-tetramethylbenzidine (TMB) is
added
a. HRP reacts with TMB (chromogenic reagent) and
gives a blue colour which can be quantified

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 iii. AntiGEN capture assay
1. Primary antibody is fixed onto the plastic wells
a. Antigen is captured by the immobilized primary
antibody
2. Captured antigen is detected by a secondary antibody,
conjugated to HRP
3. Turns the assay solution blue upon reaction with TMB
d. Goal
i. To detect if the patient has been exposed to the disease
e. Protocol
i. Antigen is bound
1. Label the 12-well strip
2. On each strip label the first 3 wells with a “+” for the
positive controls
a. Label the next 3 wells with a “–” for the negative
controls
b. Label the remaining wells to identify the samples
being tested (3 wells each)
3. Use a new pipet tip to transfer 50 μl with purified antigen
(Ag) into all 12 wells of the microplate strip
4. Wait 5 minutes for the antigen to bind to the plastic wells
5. The appropriate antigen is first absorbed to the walls of a
microplate strip
a. HIV capsid protein p24 could serve as the antigen.
ii. Wash
1. Tip the microplate strip upside down onto the paper
towels, and gently tap the strip a few times upside down
a. Make sure to avoid splashing sample back into
wells
2. Discard the top paper towel
3. Use your pipet to fill each well with wash buffer, taking
care not to spill over into neighbouring wells
a. Note: the same pipet tip is used for all washing
steps
4. Tip the microplate strip upside down onto the paper
towels and tap
a. Discard the top 2–3 paper towels
5. Repeat wash
a. Washing removes what is not bound to the
plastic.
iii. Antibody and controls
1. Use a fresh pipet tip to transfer 50 μl of the positive
control (+) into the three “+” wells
2. Use a fresh pipet tip to transfer 50 μl of the negative
control (–) into the three “–” wells
3. Transfer 50 μl of each of your team’s serum samples into
the appropriately initialed three wells
a. Use a fresh pipet tip for each serum sample

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 4. Wait 5 minutes for the antibodies to bind to their targets
a. Wash the unbound primary antibody out of the
wells two times
5. Serum that may contain antibodies against the antigen is
added to the well (human IgG)
a. If the antibodies are present in the sample, they
will bind to the antigens that are absorbed to the
wall of the well
b. If the sample contains only non-specific
antibodies, they will not bind to the antigen
6. Washing removes any antibodies which do not
specifically attach to the antigen absorbed to the well
7. Control samples (samples which will give known results)
must be run side by side with actual samples to ensure
that the assay is working correctly
iv. Secondary Antibody
1. Use a new pipet tip to transfer 50 μl of secondary
antibody (HPRO-2ndAb) into all 12 wells of the microplate
strip
a. Wait 5 minutes for the antibodies to bind to their
targets
2. Wash the unbound secondary antibody out of the wells 3
times
a. If antibodies in the previous step have attached to
the antigen, these antibodies can be detected by
the addition of an anti-human IgG
antibody-enzyme conjugate
3. The wash step gets rid of the antibody-enzyme
conjugate which did not find a specific antibody and
could not form a complex.
v. Enzyme Substrate
1. Use a new pipet tip to transfer 50 μl of enzyme substrate
(TMB) into all 12 wells of the microplate strip
a. Wait 5 minutes
2. A colour change indicates that the serum contained
specific antibodies which reacted against the original
antigen
f. SIMPLIFIED Protocol
i. Take an antigen for our pathogen of interest
1. Attach it to the bottom of plastic wells
2. The plastic has a charge that is going to attach itself to
anything we put it in contact with
ii. Wash it out
1. Put something inside to cover all the remaining exposed
plastic so it no longer attracts anything else
iii. Put in serum
1. Wash it out

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 2. If it has a primary antibody or the antigen of interest
that’s stuck on the bottom, the serum will STICK
3. If it doesn't, it won’t
iv. Add a secondary antibody
1. Attaches to the primary antibody if it is there
2. Attached to an enzyme
v. Introduce a compound
1. If it is lysed by an enzyme, the compound will change
colour
vi. If we have the antigen, primary antibody, and a secondary
antibody in the well (after the wash):
1. We will see a COLOUR CHANGE reaction (blue)
2. Indicates that we have been exposed to the disease
g. Must wash well between each of the steps (CRITICAL)
i. If we leave these components in, even if they are not specifically
attached, we will still see the colour change

/
h. Materials
i. 12-well plates
1. + controls (3)
a. Expect to turn blue
b. Have been exposed
2. - controls (3)
a. No colour change
b. Have NOT been exposed
3. Two unknown patient samples (3)
4. 3 of each to act as a safeguard
a. In case there are pipetting errors
b. Should all be the same
ii. 50 ul volumes for steps 1 to 4 will be dispensed using pipetmen
and tips

i. Antibodies
i. Primary Antibodies
1. Antibody added first
2. If testing clinical samples, 1° antibody would come from
patient serum
a. Would therefore be a human antibody
b. Have a human Fc portion
3. If the 1° antibody is specific for the antigen coating the
ELISA plate, it will bind the antigen and remain stuck to
the well
a. The 1° antibody binding site recognizes antigen
coating the plate
ii. Secondary Antibodies
1. Bind to the “heavy” chains of the 1° antibody
a. So they don’t interfere with its binding to the
antigen
b. Minimizes non-specific binding that leads to false
positives and high background noise
2. The antigen binding site of the 2° antibody has to
recognize the primary antibody

/
 a. Eg. be specific for human immunoglobulin (the 2°
antibody recognizes the Fc region)
3. The Fc region of the 2° antibody is conjugated to an
enzyme
a. HRP → horseradish peroxidase
i. An ENZYME!!
b. Enzymatically cleaves the substrate TMB
i. When this occurs, a blue colour develops
c. The intensity of the blue is a measure of the
amount of enzymatic activity
4. The secondary antibody is prepared in a species OTHER
than the one the primary antibody comes from
a. Eg. if the 1° antibody came from a human, the 2°
antibody comes from a non-human-animal source

● POST-LAB NOTES:
○ THREE Experiments:
1. Hemagglutination Assay
● Agglutination Assay
○ Determine the presence of something in solution
○ HEMA refers to RBCs being used as the source of antigen
● Blood Types (FOUR)
1. Type A
a. Anti-B antibodies
2. Type B
a. Anti-A antibodies
3. Type AB
a. NO antibodies
4. Type O
a. Anti-A antibodies
b. Anti-B antibodies
○ Agglutination occurs if for example, you gave someone
with Type B blood transfusion from a Type A
● PURPOSE:
1. To determine the PRESENCE of antibodies

/
 2. Provides a rough quantification of the AMOUNT of
antibody present in the blood (eg. titer)
● Each time, you dilute the contents by half
○ Hence, the name 2-fold dilution
● PURE SERUM (1)
○ Procedure:
■ Add saline to ALL wells
■ Pure serum (neat) is added to the 1st well
● Dilution = ½ (1 x ½)
● Take half of the solution in the first well,
and put it in the second
○ Dilution = ¼
■ Add an equal amount of blood to ALL wells
■ Incubate in fridge
■ Check if there is agglutination
○ Interpreting Results:
■ Imperfect Mat / Pellet
● First well has the highest concentration of
serum (highest amount of antibody)
○ RBCs will be SATURATED with
antibodies
○ Prevents cross-linking
(antibody-RBC)
● They are NOT binding to each other

■ Perfect mat:
● Good cross-linking
● Good antigen-antibody ratio
■ Pellet:
● Too few antibodies
○ Titer
■ INVERSE of the dilution of the last well that tests
(+) for hemagglutination (mat formation)
● Should not see any pellet...
■ eg. above is FIFTH well

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 ● Well 6 for serum 1 no longer counts as a
“positive good” because a pellet is
beginning to form
■ Find the dilution of that well and inverse it
● Impure SERUM (⅕)
○ Procedure:
■ Same process as above, except different starting
condition (⅕ x ½)
● Dilution = 1/10
■ Always multiple by ½ as you go through
○ Interpreting Results:
■ No hemagglutination
● Pellet formed in each well…
■ Was not enough antibody in the serum to begin
with
● There are too few anti-RBC antibodies
○ Titer
■ CANNOT be determined
■ No wells that show hemagglutination occur (no
mats)
2. Complement-Mediated Cytotoxicity
● Complement Cascade
○ Involves many protein substrates
■ Complement molecules
○ Complement provides EXTRA immunity:
1. Vasodilation and Chemotaxis
a. During an infection
b. Allows immune cells to easily infiltrate site
of breach
2. Opsonize pathogens
a. FLAGS them to be engulfed by phagocytes
3. Formation of the membrane attack complex
(MAC)
a. C5 - C9
b. Steps:
i. Antibody recognizes its specific
antigen
1. C1 protein binds to Ab-Ag
complex
ii. Initiates of a cascade of reactions
1. Involves C1 - C9
iii. Results in the MAC (C5-C9)
1. Lysing of cell membrane
● SIDE NOTES:
1. Complement and antibody do NOT have to come from
different species…
a. They usually don’t!!

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 i. They work in SPITE of the fact that they
are from different species
b. Complement binds to the antibody because it has
been signalled to do so
i. Since the antibody is bound to the antigen
2. Complement binds to the “constant” (Fc) region of the
antibody
a. There’s not a specific complement that binds only
to a specific Ab-Ag complex...
b. Generic complement floats around in the plasma
i. ANY Ab-Ag complex
c. Provided the antibodies are from the same class...
d. Complement is conserved across mammalian
species
3. Normal rabbit serum vs. Antiserum
a. Normal rabbit serum still has antibodies!!!!
b. Just not specific antibodies against an antigen of
interest
i. eg. sheep RBC
● FIRST PART:
○ All FIVE tubes have sheep RBCs
○ Add serum:
■ Tubes 2 & 3 → antiserum
● Has the antibody against the sheep RBC
■ Tubes 4 → normal rabbit serum
● Does not have specific sheep RBC antibody
● Still has antibodies!!
■ Tubes 1 & 5 → saline
○ Add complement to tubes 3, 4, and 5
■ None added to tubes 1 and 2
○ Only tube THREE lysed!!!
■ Only one that has both the antibody that is
SPECIFIC to the sheep RBC and the complement
system
■ Complement and antibody NEED each other to
induce lysing!!!
● SECOND PART:
○ Washed tubes 2 & 4
■ Antiserum gets washed away
■ Only antibodies that CAN attach will be left...
○ Add serum to tube 4
■ But no complement…
■ It got washed away
● Since there was no antibody for it to bind
to
○ Add complement to tube 2
■ Binds to antibodies
● Did not get washed away

/
 ■ Lysed
○ Water bath, centrifuge, and record

3. ELISA (enzyme-linked immunosorbent assay)

● Diagnostic tool
● Used to detect the presence of a specific substance
○ eg. we use it to detect the presence of a specific
antibody (in a subject’s serum) against an antigen of
interest
● Procedure:
○ Bottom of wells have (-) charge
■ So substrate (eg. antigen) sticks
○ Add antigen of interest to all the wells
■ Allow for them to bind
■ Wash away any that didn’t
○ Add sera:
■ (+)
● Binded
● Knew had the antibodies specific to the
antigen
● eg. patinet A
■ (-)
● Nothing binded
● Knew it did NOT have the antibodies
● eg. patient B
■ Wash
● Get rid of any antibody that didn’t bind
○ Added a secondary antibody
■ Recognizes the Fc region (constant) of the
primary antibody
● Will recognize and bind to it in (+) cases
■ Conjugated to an enzyme called: horseradish
peroxidase
■ Wash
● Get rid of any antibody that didn’t bind
○ Add substrate (TMB)
■ If the secondary antibody was present
● If the primary antibody was present…
■ The enzyme on the secondary antibody CLEAVES
TMB

/
 ● Releases a blue colour in (+) wells
■ No colour in (-) wells since the secondary
antibodies got washed away…

● SIDE NOTES:
○ In order to produce secondary antibodies…
■ Need to recognize the first antibody as FOREIGN
○ So, the secondary antibody must come from a
DIFFERENT species
■ If it were from the same species, it wouldn’t
recognize the primary antibody as being “foreign”
○ Testing (+) for an antibody does NOT imply that you have
been exposed to the disease...
■ Reasons for testing (+):
● Past exposure to the antigen-relevant
disease
● Immunization via a vaccine
○ Note that getting a vaccine is NOT
the same as getting the disease
○ Can contain just antigen fragments,
etc...
● Receive antibodies from the mother
○ Through the placenta or through
breast milk

/
2. BLOOD
● Overview
○ Will be given blood from one of 3 clinical cases
○ Work out which blood you have based on set of experiments
● Basic Concepts:
○ Blood
■ A CRITICAL physiological tissue
■ Functions
● Nutrient and oxygen transport
● Metabolic water removal
● Distributing heat
● Maintaining pH
● Immune defense
● Wound healing
● Tissue-to-tissue communication (hormones)
● Pathogen dissemination/transmission
■ Composition
1. Plasma
a. Mostly water
b. Some plasma proteins
2. Cells
a. Mainly RBCs
b. Some WBCs (leukocytes)
3. Cell Fragments
a. Eg. platelets
■ Practical Uses
● Diagnostics
● Life-saving transfusions
● Erythrocyte Fragility and Osmosis
○ RBCs will rupture (hemolyse) when subjected to
osmotic stress
■ Cell contents will be released into solution
(hemoglobin)
○ The osmotic fragility test is designed to:
■ Give some information on the capacity of
the red cell membrane to withstand
increasing internal pressures
■ Brought about by the diffusion of water into the cell
○ Osmosis
■ Net diffusion of water across a membrane
■ Due to a concentration difference
● Results in a net flux
● To reestablish the same water concentration on both sides
○ Adding solute to water will DECREASE the concentration of water

/
 ■ The degree as to which the concentration of water is reduced by a
solute depends on the NUMBER OF PARTICLES of the solute in solution
■ Osmolarity
● Measure of the solute concentration of a solution
○ The amount of osmotic pressure depends upon the difference between the
concentration of non-diffusible ions (osmoles/L) on each side of the
membrane
■ One osmol is equal to one mol of dissolved, non-diffusible,
non-ionizable substance
● If the substance completely ionizes into two ions, then one mol
of the dissolved substance yields two osmoles
■ 1 osmol = 1 mol of solute
● Eg. NaCl = 2 Osmoles (1 Na + 1 Cl)
○ Flow of water will be from LOW osmolarity solution to HIGH osmolarity
solution

○ Cells have semi-permeable membranes

■ Permeable to water
● But, some solutes are actively kept inside the cell
■ Thus, in order to achieve equilibrium in water concentration on both
sides of the membrane, the side with the greater osmolarity will GAIN
volume
○ Osmotic Pressure
■ The pressure that must be applied to the solution to PREVENT the net
flow of water
1. Isotonic Solution
a. No change in cell volume
b. Osmolarity = 300 mOsm = 0.3 Osm
c. A 0.9% NaCl (MW = 58.5) solution
i. Intracellular and extracellular fluids are in osmotic
equilibrium across the cell membrane
ii. No net influx or efflux of water
2. Hypertonic Solution
a. Cell shrinks (loses water)
b. Crenation
i. Cells collapse
c. e.g. 1.8% NaCl

/
 3. Hypotonic Solution
a. Cell swells
b. Hemolysis
i. Swells to a point that the membrane bursts
ii. The integrity of their membranes is disrupted, allowing
the escape of their hemoglobin which dissolves in the
external medium.
c. e.g. 0.4% NaCl or distilled water

○ Calculations

■ Eg.

○ Spectrophotometry
■ Measures the quantity of light absorbed by particles in that solution
when a light is shone through it
● Optimum wavelength for absorption by Hb = 540 nm
■ Optical Density (OD)
● Directly proportional to the # of solute particles in the solution
● MORE PARTICLES = MORE
ABSORBANCE
● Increases linearly with concentration
of Hb in solution

/
 ○ Amount of Hb in solution depends on the # of RBCs that
have hemolyzed
● Erythrocyte Sedimentation Rate (ESR)
○ A non-specific assessment of inflammation
■ By recording effects of plasma proteins on rate of settling out of RBCs
from plasma
○ Principle
■ RBCs are denser than plasma and will settle under the influence of
gravity
■ The rate at which they settle depends on fibrinogen and globulin
concentrations
● These make RBCs stick together to form stacks of cells called
rouleaux
■ Rouleaux settle more rapidly than individual cells (high ESR)
● These concentrations INCREASE with inflammation
● Blood Cell Indices
1. Hematocrit (Ht)
a. % of blood volume occupied by RBCs
b. Anemia
i. Low RBC production
ii. High RBC destruction
iii. Small RBC size
c. Polycythemia
i. Excess RBC production
ii. Larger RBC size

2. Hemoglobin Content Determination

a. Hemoglobinometer
i. Compares the colour of light passing through
a hemolyzed blood sample with a standard
colour

/
 1. Spectrophotometric measurement of hemoglobin
concentration in a hemolyzed sample
ii. Readout is grams of hemoglobin per 100 mL of blood
b. Normal range = 12 - 18

3. RBC Count
a. Hemocytometer
i. Used to count cells

b. Mean Corpuscular Volume (MCV)

i. Average volume of RBC

c. Mean Corpuscular Hemoglobin Concentration (MCHC)

i. Average hemoglobin concentration per unit volume (100 mL) of
packed RBC

/
4. WBC Count

a. Neutrophil
i. MORE than 2 lobes in nucleus
b. Eosinophil
i. 2 lobes in the nucleus
ii. More granular → more colourful
c. Basophil
i. Packed with granules
d. Lymphocytes
i. Nucleus takes up most of the cell
ii. Almost entirely blue
e. Monocytes
i. Kidney-shaped nucleus
1. Does not take up the entire cytoplasm
ii. Looks similar to a lymphocyte

5. Blood Group Typing

a. ABO
i. ABO human blood group antigens

/
 ii. First discovered by Karl Landsteiner
1. 1901
2. Austrian
3. Enabled successful blood transfusions
a. 1930 Nobel Prize
iii. 4 blood groups determined by the presence or
absence of specific antigens on the RBC surface

b. Antibodies (agglutinins) for antigens A and B exist in the plasma and

these are termed anti-A and anti-B
i. The corresponding antigen and antibody are never found in the
same individual since, when mixed, they form antigen-antibody
complexes, effectively agglutinating the blood.
c. Antigens → cell surface proteins
i. Agglutinins → antibodies against RBC antigens
d. What happens when the wrong blood is put into a patient?
i. Agglutination
1. Clumping of particles
ii. Occurs when blood is transfused into a recipient who has
agglutinins (opposing antibodies) against the donor’s
agglutinogens (RBC target antigens)
iii. Transfusion Reaction
1. Destruction of transfused cells by recipient
2. Eg. fever, chills, fainting, dizziness
a. Acute immune response

/
 e. Rh
i. More than 40 antigens with RH0 being the most likely to cause a
problem
ii. Can either have the antigen (Rh+) or lack the antigen (Rh-)
iii. Most people are Rh+

iv. Sensitization
1. An Rh- individual can produce anti-Rh agglutinins upon
exposure to Rh+ blood
2. Can cause problems during pregnancy
a. If mother is Rh+, then it does not matter what the
baby is
3. If the mother is Rh-, and if the baby is Rh+:
a. In the first pregnancy, problems seldom develop
as very few fetal cells cross into maternal
circulation
b. However, at birth:
i. Blood mixing causes mother’s immune
system to produce anti-Rh agglutinins
(sensitization)
ii. Takes a few weeks so first baby is not
affected, but future babies would be!
c. In the secondary pregnancy, agglutinogens of
mother agglutinate fetal RBCs and cause
hemolysis (hemolytic disease)
i. This can cause severe anemia and the
death of the fetus
● POST-LAB NOTES:
○ Experimental Error
■ Errors that may occur in the execution of an experiment design
1. Not mixing blood
2. Making incorrect dilutions
3. Not taking measurements at the right time
■ NOT

/
 1. Setup of ESR tubes
2. Differences in room temperature
3. Calibration of spectrophotometer
1. Erythrocyte Fragility
■ RBCs undergo changes in solutions of different tonicity
● Due to osmosis (osmotic pressure)
● From the efflux or influx of water out of or into the cell

■ Procedure:
● Add RBCs to different solutions of NaCl
● Undergo centrifugation
○ In water, all the cells will lyse
■ Hemoglobin makes it red
○ In 0.9% NaCl (isotonic solution), the cells will form a
pellet at the bottom of the tube
● Measure with spectrophotometer
● Common Errors:
○ Adding wrong volume of blood to the NaCl solutions
■ eg. 5 vs. 50 µL
○ Slightly resuspending pellet after centrifugation

● Plotted onto curve

○ Osmolarity vs. % Hemolysis
○ The second point from the right has still not lysed…

/
 ■ But, the cell is swelling!!
● Just hasn’t burst yet
■ Cells are resistant until a certain point
● Some limit to what they can handle…
○ After this point, the RBCs all rapidly hemolyse
■ To almost 100%
○ RIGHT vs. WRONG

○ eg. Anemic
■ Curve shifted to RIGHT
● RBCs lyse closer to
isotonicity
● More fragile!
2. Erythrocyte Sedimentation Rate (ESR)
■ RBCs are denser than plasma
● So they will settle over time
■ Measure of the rate of mm of plasma over an hour
■ eg. gives you information whether or not there is
inflammation or tissue damage
● Would INCREASE the packing of the RBCs →
greater ESR
■ Common Errors:
● Not reading the ESR one hour after it was
started
● If reading late, trying to back-calculate what
the ESR would have been at 1 hour
○ Not accurate
○ We don’t know the kinetics of the rate…
3. Hemostatic Test
■ One patient had an INCREASED bleeding time
● Associated with reduced platelet counts
4. Blood Cell Indices
■ Tests
1. Hematocrit
2. Hemoglobin Concentration
3. RBC/WBC Counts
a. Common Errors:

/
 i. Mixing
ii. Pipetting
iii. Counting errors
1. Black (correct) vs. red (incorrect)

4. Differential Blood Stain

a. Different blood cell types
b. Common Errors:
i. Mistaking lymphocytes for basophils (granule)
1. Differentiate based on granularity
ii. Mistaking neutrophils for eosinophils
1. Differentiate based on # of lobes
c. One patient had a LOT of lymphocytes
i. Healthy vs. Sick

5. ABO Blood Group Typing

■ But different antibodies on the blood
● Anti-B, Anti-A, Anti-Rh
■ Checked for agglutination
■ Common Errors:
● Calling something not agglutinated when it was..

○ CBC Report

/
 ■ Derived too many conclusions from the MCV and MCHC, when other
primary values were normal…
● Treat these as secondary values
● Can give the exact “flavour” of the disease
○ Cases:
■ Each group received blood from one of three clinical cases
● Worked out which blood you had based on the data
■ CASE 1 → ANEMIA
● All point towards autoimmune hemolytic anemia
● Signs:
○ Decreased RBC count
■ Decreased hematocrit
■ Decreased hemoglobin
○ Fragile cells
■ Erythrocyte fragility curve shifted to right
○ Elevated ESR
■ More inflammation...
● Causes:
1. Excessive RBC loss
a. Blood loss
b. Increased RBC destruction (hemolysis)
i. Extrinsic
1. Infection
2. Medications
ii. Intrinsic
1. Autoimmune hemolytic anemia
2. Inadequate RBC production
a. Iron deficiency
b. Folic acid deficiency
● Common Mistakes:

/
 ○ Not having erythrocyte fragility curve shifted
■ Not mixing blood well
■ Not pipetting correct volume of blood
■ Disturbing the pellet after centrifugation
○ Not connecting what increased ESR has to do with
anemia
■ Destruction of RBCs

■ Case 2 → FEVER
● Signs:
○ Increased WBC count
■ Infection
○ Increased ESR
■ Inflammation
○ % of neutrophils and eosinophils
■ Parasite or bacteria
● Common Errors:
○ Errors in WBC total count and differential count
■ Making it difficult to form a hypothesis
○ Overinterpreting small changes in RBC number or
MCV/MCHC

/
■ Case 3 → LEUKEMIA
● Signs:
○ Increased WBC count
○ High # of lymphocytes
○ Increased bleeding time (low platelets)
■ Results in bruising easily
● “Pregnancy” feeling was supposed to be ignored lol
● Types of Leukemia/Blood Cancers:

○ Stem from bone marrow

○ Results in:
■ Cancers
● Usually arising from bone marrow
● Leading to high numbers of abnormal
WBCs
■ High risk of bleeding
● Reduced platelets
○ Cancer cells take up all the
nutrients in the bone marrow
● Deprives other cells of the nutrients they
need to mature
■ High risk of infection
● Since the WBC are not acting normally
● Common Mistake:
○ Not correlating extremely high lymphocyte count to a
proliferative disorder (cancer)
○ Not correlating
increased bleeding time
with bruising
○ Not taking into account
that none of the
inflammatory markers
(CRP, ESR) are elevated
■ UNLIKELY that it
is an infection

/
○ Practice Questions:

■ Sum of counts
● 95 + 82
■ Dilution factor
● Putting 20 µL into 80 µL = 100 µL
● 100 / 20 = 5
■ # squares = 2 (x 0.1)

■ D is technically right, but is not the best answer

● Has to be a hypotonic solution
■ C can be directly inferred from the information given

■ Decreased ESR → less inflammation

■ Increased RBC count → N/A
■ Decreased hemoglobin content → N/A
■ High CRP → more inflammation

/
 ■ Measuring hemoglobin and hematocrit will tell you if the patient is
anemic
5. If you have a WBC count that is VERY HIGH, what other value(s) could you
corroborate that it is in fact high?
■ Usually when there is an infection
■ Check if ESR and CRP are HIGH

/
3. BIOMEDICAL SIGNAL ACQUISITION
● Purpose:
1. Understand the concepts and practical issues of how to properly acquire a
physiological signal:
a. Computer-based data acquisition
b. Digital signal processing
2. Understand the sources of interference that can affect recorded signals
a. Understand how to reduce them
3. Understand different ways to analyze data
4. Measure human EEG
5. Understand the basic concepts
● Concept Map:

● THREE Experiments:
1. Waveform Acquisition
a. What is a waveform?
b. How do you record it properly?

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 2. Artifacts Contaminating the EEG Recordings
a. Understand limitations of the recording equipment
b. How do you reduce artifacts?
i. Artifacts
1. Distortions in the signal of interest due to OTHER
physiological processes in the body
c. Not using a biological signal…
i. Start with a function generator → puts out a sine wave
ii. Change the amplitude and frequency
3. Alpha Waves in the EEG
a. Measure brain waves in subject
● SIGNAL ACQUISITION
○ Signal
■ A coded message sent from one organism to another containing
information
● OR from one place in an organism to another place
■ Typically contain information about the behavior or nature of some
phenomenon

■ Must be able to decode it…

● Use MATH to describe it:
○ Function of one or more independent variables
● F(x1, x2, xn) that contain information about the behaviour of
some phenomenon
■ Systems usually respond to particular signals by producing other signals
● Electrical brain activity (signal) => Power Lab (system) =>
voltage (signal) variations in time
■ Information in a signal is contained in a pattern of variations of some
form
● eg. the human vocal mechanism produces speech by creating
fluctuations in acoustic pressure
● Electrophysiology
○ Measures the flow of ions in biological tissues and the related changes in
potential
○ Recording electrical signals

○ INTRAcellular Potentials
■ Not going to be using this
○ EXTRAcellular Potentials
■ EKG/ECG
● From heart

/
 ■ EEG
● Put electrodes on the scalp or our subject
○ Measure potentials OUTSIDE the skull
● Shape of waveform is inverted (from the picture above):
○ Measuring currents flowing in to the neuron
○ Rather than the change in the membrane potential
■ Spirogram
● From lungs

○ TYPES of Signals:
■ Deterministic Signals
● If a signal is exactly predictable for the time span of interest
○ Can be described by mathematical models
● E.g.
○ A sinusoidal signal is described by:
○ V(t) = A * sin(ω*t)
■ V(t) is the signal over time
■ ‘A’ is the amplitude
■ ω= 2f
● f= frequency of the signal
■ Stochastic / Random Signals
● A signal whose value has some element of chance associated
with it
○ Therefore, it CANNOT be predicted exactly by a
mathematical equation
● Consequently, statistical properties and probabilities must be
used to describe stochastic signals
■ Usually, biological signals often have both deterministic and stochastic
components
■ TIME Signals:
● ANALOG / Continuous time signals
○ The independent variable is continuous
■ The signals are defined for a continuum of values
of the independent variable X(t)
● DIGITAL / Discrete time signals
○ Only defined at discrete times

/
 ■ The independent variable takes on only a discrete
set of values X(n)
○ May represent a phenomenon for which the independent
variable is inherently discrete
■ eg.
● Amount of calories per day on a diet
● An analog signal is a continuous time
signal
○ On the other hand, it may represent successive samples
of an underlying phenomenon for which the independent
variable is continuous
■ Eg.
● Visual image captured by a digital camera
is made of individual pixels that can
assume different colors
● A digital signal is a discrete time signal
○ Important to take ENOUGH POINTS to accurate depict
the signal
○ Signal CHARACTERISTICS
■ Period
● Distance between two peaks
■ Frequency
● The number of oscillations in one second
● It is measured in Hertz (Hz)
○ eg. 1 Hz = 1 oscillation/cycle in a period of 1 second
● eg.
○ The alternating current in a wall outlet in the U.S. and
Canada is 60Hz
■ Amplitude
● Wave form from axis to peak
● Maximal and minimal amplitude
○ Maximal and minimal value of the signal during a given
time interval
● Range
○ Peak-to-peak amplitude
○ The difference between the minimum and maximum
values of a signal
○ Waveform going up + waveform going down
● Statistics
○ A number of statistics may be used as a measure of the location or
“center" of a random signal:
a. Mean
i. Average amplitude of the signal over time
b. Median
i. Value at which half of the observations in the sample
have values SMALLER than the median
1. Other half have values LARGER than the median

/
 ii. The median is often used as a measure of the “center" of
a signal
1. Less sensitive to outliers
c. Mode
i. Most frequently occurring value of the signal
ii. Maximal and Minimal amplitude
1. The maximal and minimal maximal values of the
signal during a given time interval
■ Waveform
● The representation of a signal as a plot of signal amplitude
versus time

○ Spectral Analysis
■ The process of decomposing a signal into DIFFERENT frequency
components
● Plot the intensity of each component as a function of its
frequency
■ Fourier / Power Spectral Density (PSD) Analysis
● A biological signal can be broken down into fundamental
frequencies
○ Each frequency has its own intensity
● Display of the intensities at all frequencies
● It is a mathematical technique that allows us to perform a
spectral analysis on a recorded signal
○ Separate the signal
○ Desired Signal
■ A signal that it is NOT corrupted by noise
○ NOISE
■ Any UNWANTED signal that modifies the desired signal
● It can have multiple sources
■ Types:
1. Thermal
a. The random motion of atoms generates this random,
uniformly distributed noise → “white noise”
i. Present everywhere
b. Has a nearly constant Power Spectral Density (PSD)
2. Interference

/
 a. Imposition of an unwanted signal from an external
source on the signal of interest
3. Sampling
a. Occurs when you digitize a continuous signal with an A/D
converter that has a finite number of steps
b. You can dither (add white noise: eg. does not vary with
frequency) your signal to reduce the overall sampling
noise
● Categories:
i. Two general categories of noise
ii. NARROWband Noise
1. Confines itself to a relatively SMALL portion of the overall
signal bandwidth as defined by Nyquist
2. eg.
a. 60Hz-hum is narrowband
b. Because it typically limits itself to a 60 Hz
component
iii. BROADband Noise
1. Occupies a SIGNIFICANT portion of the Nyquist
bandwidth
2. eg.
a. Thermal noise is definitely broadband
b. Because its PSD is constant
i. Meaning that it distributes its energy over
nearly the entire spectrum
■ Signal to Noise Ratio (SNR)
● It is a measurement of the amplitude of variance of the signal
relative to the variance of the noise
● The higher the SNR, the better you can distinguish your signal
from the noise
○ How do we measure biological signals in the lab?
■ Electrode or sensor
● Picks up the electric potentials from the cell or subject
■ Convert the measured signal into something that we can record and
process on a computer
● Biological signals tend to be continuous
○ We must turn the continuous signal into a discrete
signal!!
● Take points at different times
○ To represent the waveform in the computer
■ TRANSDUCER
● Converts the analogue (non-voltage) signal into an analogue
voltage signal
○ Since the ADC is an electronic device, it requires an
electrical signal at its input
● Not necessary if signals are inherently voltages
○ eg.
■ Electrocardiogram (heart)

/
 ■ Electrooculogram (eyes)
■ Electromyogram (muscles)
■ Analog-Digital Converters (ADC)
● Converts ANALOG signals into DIGITAL signals
○ Allows it to be analyzed by a computer to visualize it on
the screen
● We are using a 16-bit board:
○ This means that when it performs an A/D conversion, the
ADC samples the analogue voltage present at its input at
that point in time and converts it into a 16-digit binary
number
■ Since each digit of a binary number can take one
of two values 0 or 1:
● A 16 bit (bit = binary digit) number can
take one of 2^16 = 65536 values
● Representing the integers from 0 to 65535
○ Has an input range of -10 000 millivolts (mV) to +10 000
millivolts (mV)
■ Binary = 0000000000000000 (base 2)
● Decimal = 0 (base 10)
● Will be returned by the ADC to the
computer when a voltage of -10 000 mV
is present at its input
■ Binary = 1111111111111111 (base 2)
● Decimal = 65535 (base 10)
● Will be returned when the input voltage is
+10 000 mV
○ Thus, the input voltage range from -10 000 mV to +10
000 mV is divided into 65536 levels
■ Each level being 20 000 mV / 65536 = 0.305 mV
wide
● This value will determine the resolution of the sampled signal

● High vs. Low Resolution:

○ High
■ Levels would be invisible
○ Low
■ levels/steps are MUCH clearer
● How would the resolution for an 8-bit converter compare to a
12-bit case?
○ The resolution of an 8-bit converter is NOT as good as
that of a 12-bit one
■ 12-bit converter resolution = 0.0049 volts
■ 8-bit converter resolution = 0.078 volts
● Not able to resolve the smaller, finer
changes in the input signal

/
 ● Therefore, the sampled signal will not be
as accurate a representation of the true
signal
● It is important to keep the input signal WITHIN the input voltage
range of the ADC
○ If the input voltage exceeds the ±10 volt range, a 16-bit
binary number with an equivalent decimal value of
65535 is still returned to the computer
■ The computer would thus interpret the voltage
being sensed to be +10 000 mV, which would be
an error
○ This error is called SATURATION
■ When the intensity of a signal EXCEEDS the
values within the sampling range
○ eg.
■ If we acquire a signal which intensity is +20V and
we are sampling between -5V and +5V. It
produces a distortion of the signal
■ Over the interval in which the signal reaches the
+20V, the output of our ADC will be +5V
● However, the input signal to the ADC should also SPAN as much
of the ADC input voltage range as possible, without saturating
the ADC
○ This increases the signal resolution
■ Signal to noise ratio
■ Thus, if the voltage range of the input signal is
much smaller than +/- 10 000 mV:
● The signal should be AMPLIFIED before
being fed to the input of the APC
○ eg.
■ If the signal to be recorded is much smaller than
±10 000 mV, say ±5 000 mV, then the range
over which the board operates should be
decreased
● By changing the hardware gain from 10
000 mV (10 V) to 5 V, the operating range
of the board is changed from ±10 000 mV
to ±5 000 mV
■ This allows the experimenter to record the ±2 V
signal with a significant improvement in signal
resolution (2 times greater)
● This occurs because the minimum
resolvable voltage would be 10
000mV/65535 or 0.152 mV versus 0.305
mV when the board's operating range was
set to ±10 volts
■ To INCREASE the size of the signal or waveform to be measured more
appropriately, we use an:

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 ● AMPLIFIER
○ A device that tracks the amplitude of an incoming signal
○ Proportionally increases the voltage, current or power of
the signal by using power from another source (plug into
the wall)
■ DOES NOT CHANGE THE FREQUENCY, only
changes the amplitude
○ Multiplies the signal
■ GAIN
● The factor by which the amplifier
multiplies the signal (waveform)
○ If gain = 1, the signal does not
change
○ If gain > 1, the signal is amplified
○ If gain < 1, the signal is reduced
○ eg. power lab

○ But, amplifying the signal ALSO amplifies the noise…

■ How do we get rid of noise?
1. Single-Ended Amplifier
a. Keeps two inputs constant
i. Output reads the difference
b. Not very helpful, also amplifies the noise...
2. DIFFERENTIAL Amplifier
a. It is the key to electrophysiological equipment
i. It magnifies the difference between two inputs
b. Idea:
i. An unwanted signal that is common to the two
inputs will be subtracted
1. Noise should be the SAME at both
electrodes
a. Signal should NOT be the same
2. So, subtract out whatever is the “same”
a. Amplify the difference (what is left
over)
ii. Results in only the amplification of the signal
c. PROCESS:

/
 i. Two active inputs
1. Recording from two locations
ii. Compare the two inputs
1. Subtract the two inputs
2. Amplify the difference

○ Signal Filtering:
■ Usually we are interested in signals of a PARTICULAR frequency
bandwidth (range)
■ The bandwidth is determined by:
● Filters
○ A device or circuit which permits CERTAIN frequency
components of a signal to pass easily while inhibiting or
preventing others
■ It is typically used in physiology to separate the
desired Signal from Noise
● Ideal vs. Non-Ideal
○ IDEAL
■ COMPLETELY eliminates all frequencies above the
cut-off frequency
● While passing those below unchanged
■ How is it done?
● By reducing the amplitude of frequency
signals above the cut-off frequency
■ Provides a very sharp transition between the
frequencies that are passed and those that are
filtered out
■ This in practice CANNOT be achieved
○ NON-IDEAL
■ Passes low-frequency signals

/
 ■ BUT attenuates (reduces the amplitude of) signals
with frequencies higher than the cut-off
frequencies
○ METHODS of Filtering:
1. Band Pass / Ideal Frequency-Selective Filter
a. Allows only signals within the specified range to pass
through the filter, and rejects the rest
i. Useful when you want to retain only specific
waves from an EEG record
b. eg.
i. Retain alpha waves
ii. Can set the filter to only pass data between 8Hz
and 13Hz
c. Low Pass (High Frequency) Filter
i. Block high frequencies, and let low frequencies
pass
ii. Attenuates (reduces) the signals ABOVE a given
threshold
iii. eg.
1. Any ADC has a maximum sampling rate
2. In some circumstances, this maximum
sampling rate is NOT high enough to
satisfy the Nyquist conditions
3. In that case, one can pass the analogue
signal through a low-pass filter before
sending it on to the ADC
4. This filter acts to remove some of the
high-frequency content of the signal
a. Would otherwise alias down in
frequency
b. Producing spurious low-frequency
content
5. Note that this anti-alias filtering could
remove high frequency information of
physiological importance to the
phenomenon under investigation
a. If it is important to retain these
higher frequencies, one has no
choice but to use a better data
acquisition system that has a
higher sampling rate
d. High Pass (Low Frequency) Filter
i. Block low frequencies, and let high frequencies
pass
e. Notch Filter
i. Blocks a specific frequency
2. Usually 60 Hz (noise)
○ TYPES of Filtering:

/
 1. Hardware (REAL) Filtering
a. A component of the amplifier
i. Alters the frequency composition of the signal
b. Happens BEFORE we digitize the signal (send it to the
computer)
i. Means after filtering the signal, we CANNOT
recover the frequencies that have been filtered
2. Digital Filtering
a. Done (by software) by the computer
i. Change the frequency of the signal by performing
calculations on the data
b. It means you CAN record all the frequency components
of your signal
i. By digitally filtering it, eliminate the unwanted
frequencies
c. You can still recover the filtered frequencies if you keep a
record of the original signal
○ Other important ARTIFACTS:
■ Signal Offset
● A constant and steady
deviation of the measured
signal from the set point
○ In other words, a
fluctuation in the
baseline value of
the signal
● There are usually offset
buttons where you can
move the signal back to
the central position
● Not a problem because we are usually not interested in signals
that do not change
○ We’re interested in HOW signals change OVER TIME
■ Saturation
● When there is too large of an offset
● When you go BEYOND the range for which the recordings can
be made
○ Rather than being able to measure the entire sine wave,
the peaks are CLIPPED OFF
○ Instead of actually measuring the change, you’d be
reading off the peak
value instead of
reading the actual
value
● eg.
○ Setting a GAIN (that
could reduce the
amplitude of the

/
 waveform) would be useful
■ Signal Sampling
● The process of obtaining a sequence of instantaneous values of
a particular signal characteristic
● Usually at regular time intervals
● Sampling Frequency
○ Number of points (samples) per second (Hz)
○ The frequency at which the ADC samples the analogue
signal
● Sampling Period
○ The reciprocal of the sampling frequency
○ eg. the interval between corresponding points on two
successive sampling pulses of the sampling signal
● Sampling Range
○ The range between the minimal and maximal values at
which you will sample the signal
■ eg. If you sample between -10 V and +10 V the
sampling range is 20 V
■ ALIASING
● When NOT enough samples are taken of the digital time signals
○ Causes a high frequency signal to appear to be of a
lower frequency
● Occurs when the following rule is NOT followed: NYQUIST
○ Sampling Interval
■ The inverse of the sampling rate
■ The MAXIMUM time interval between equally
spaced samples of a signal that will enable the
signal waveform to be completely recovered
○ Sampling Rate
■ It is equal to twice the highest frequency
component of the sampled signal
● # of samples = 2x the maximum
frequency
■ Eg. 5 Hz sine wave requires more than 10 points
to reproduce the sine wave
● The faster the rate at which a signal
changes
● The higher the frequency content of the
signal
● The higher the sampling rate needed to
reproduce it faithfully
■ The required sampling rate is determined by the
accuracy of the digitizing process
■ Important that we set our sampling rate
APPROPRIATELY
■ What happens if we OVERSAMPLE?
○ You want to find a balance between being
able to reproduce the signal (fidelity of

/
 reproduction), but not increasing the
processing time (computer storage space,
computing time, cost)
1. For a given frequency content, increasing
the sampling rate beyond a certain point
does NOT significantly increase the
accuracy with which the signal is rendered
2. Cost of an ADC increases as higher
sampling rates are desired
3. More computer processing time
and storage space in memory or
disk are needed to process the
larger number of data points
produced when the sampling rate is
increased
○ Sampling Theory
■ In practice, when analog signals are sampled for
the purpose of digital processing:
● The sampling rate must be MORE frequent
than that defined by the Nyquist interval
■ Because of quantization error introduced by the
digitizing process
● To provide a safety factor to guard against
information loss:
■ It is usual to sample at 5-10 times the highest
expected frequency
● RATHER than the minimum two times

● Electroencephalogram (EEG)
○ A noninvasive, diagnostic technique that records electrical impulses produced
by brain cell activity
■ The recorded waveforms reflect the cortical electrical activity
○ Some of the recorded activity is generated by action potentials
■ But MOST is generated by excitatory and inhibitory postsynaptic
potentials (EPSPs and IPSPs)
○ Measure it by putting electrodes on our scalp, along with electro paste
■ The paste INCREASES the
contact
○ Differential Amplification
○ Pathway:

/
 1. Reference and active electrodes
a. Amplify it
2. Send it to the A-D Converter
3. Montage Generator
4. Filter
5. Digital Display
○ EEG systems have come a long way…
■ Used to be something that takes up half a room
● Now, it is miniaturized (can take it anywhere)
○ Often used in sleep studies, and epilepsy:
■ Can find the regions of the brain where seizures originate from
■ Can also look at the frequency to see what kind of seizures they are
● Eg. epilepsy (absence seizures) vs. primary generalized epilepsy

○ Signal
■ Intensity
● EEG activity is quite small
● Measured in microvolts (µV)
■ Frequency
● The main frequencies of the human EEG waves are (in order of
increasing frequency/decreasing amplitude):

● Delta
○ Frequency of 3 Hz or below
■ HIGHEST in amplitude
■ SLOWEST waves
○ Normal:
■ As the dominant rhythm in infants up to one year
■ In stages 3 and 4 of sleep

/
 ○ It may occur focally with subcortical lesions and in
general distribution with diffuse lesions, metabolic
encephalopathy hydrocephalus or deep midline lesions
○ Most prominent:
■ Frontally in adults
● e.g. FIRDA - Frontal Intermittent Rhythmic
Delta
■ Posteriorly in children
● e.g. OIRDA - Occipital Intermittent
Rhythmic Delta
● Theta
○ Frequency of 3.5 to 7.5 Hz
■ "slow" activity
○ Normal in:
■ Children up to 13 years
■ In sleep
○ Abnormal in:
■ Awake adults
■ It can be seen as a manifestation of focal
subcortical lesions
● It can also be seen in generalized
distribution in diffuse disorders
● eg.
○ Metabolic encephalopathy
■ Some instances of hydrocephalus
● Alpha
○ Frequency between 7.5 and 13 Hz
■ Is usually best seen in the posterior regions of
the head on each side, being higher in amplitude
on the dominant side
○ It appears when closing the eyes and relaxing
■ It disappears when opening the eyes or alerting
by any mechanism:
● Thinking
● Calculating
● etc...
○ It is the major rhythm seen in normal relaxed adults
○ It is present during most of life especially after the
thirteenth year
● Beta
○ Frequency of 14 Hz and up to 20 Hz
■ 'FAST' activity
○ It is generally regarded as a normal rhythm
■ It is the dominant rhythm in patients who:
● Are alert
● Are anxious
● Have their eyes open

/
 ○ It is usually seen on both sides in symmetrical
distribution
■ Most evident frontally
○ It is accentuated by sedative-hypnotic drugs
■ Especially benzodiazepines and barbiturates
○ It may be absent or reduced in areas of cortical damage
○ VARIABLES
■ Frequency
● The rhythmic repetitive EEG activity (in Hz)
● The frequency of EEG activity can have different properties
including:
a. Rhythmic
i. EEG activity consisting of waves of approximately
constant frequency
b. ARrhythmic
i. EEG activity in which no stable rhythms are
present
c. DYSrhythmic
i. Rhythms and/or patterns of EEG activity that
characteristically appear in patient groups or can
be rarely seen in healthy subjects
■ Voltage
● The average voltage or peak voltage of EEG activity
○ Values are dependent, in part, on the recording
technique
● Descriptive Terms:
1. Attenuation
a. Reduction of amplitude of EEG activity resulting
from decreased voltage
i. Suppression, depression
b. When activity is attenuated by stimulation, it is
said to have been "blocked" or to show “blocking”
2. Hypersynchrony
a. An increase in voltage and regularity of rhythmic
activity
i. Often within the alpha, beta or theta
range
b. Implies an increase in the number of neural
elements contributing to the rhythm
c. The term is not used in interpretative sense, but
as DESCRIPTOR of change in the EEG
3. Paroxysmal
a. Activity that emerges from background with a
rapid onset
i. Usually reaches quite a high voltage
1. Ends with an abrupt return to lower
voltage activity

/
 b. Though the term does NOT directly imply
abnormality, much abnormal activity is
paroxysmal
■ Morphology
● The shape of the waveform
● Determined by the:
○ Frequencies that combine to make up the waveform
○ Phase and voltage relationships
● Wave patterns can be described as being:
1. Monomorphic
a. Distinct EEG activity appearing to be composed of
one dominant activity
2. Polymorphic
a. Distinct EEG activity composed of multiple
frequencies that combine to form a complex
waveform
3. Sinusoidal
a. Waves resembling sine waves
b. Monomorphic activity usually is sinusoidal
4. Transient
a. An isolated wave or pattern that is distinctly
different from background activity
b. A transient with a pointed peak
i. Spike
1. Duration from 20 to under 70 msec
ii. Sharp Wave
1. Duration of 70-200 msec
■ Synchrony
● The simultaneous appearance of rhythmic or morphologically
distinct patterns
● Over different regions of the head:
○ Either on the:
■ Same side (unilateral)
■ Both sides (bilateral)
■ Periodicity
● The distribution of patterns or elements in time
● eg.
○ The appearance of a particular EEG activity at more or
less regular intervals
● The activity may be generalized, focal, or lateralized
■ EEG Electrodes
● Small metal discs placed on the scalp in special positions
○ Usually made of stainless steel, tin, gold or silver
○ Covered with a silver chloride coating
● These positions are specified using the International:
○ 10/20 System
● Each electrode site is labeled with a:
1. Letter

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 a. Refers to the area of the brain
underlying the electrode
b. eg.
i. F - Frontal lobe
ii. T - Temporal lobe
2. Number
a. Even
i. Right side of the head
b. Odd
i. Left side of the head
● Electrodes used in EEG recording DO NOT discriminate the
electrical signals they receive
○ The recorded activity which is not of cerebral origin is
termed an artifact
○ Can be divided into:
■ Physiologic Artifacts
● Generated from the subject from sources
other than the brain
■ Extraphysiologic Artifacts
● Arise from outside the body
● Equipment including the electrodes and
the environment
■ Electrode Gel
● Maximizes skin contact
○ Allows for a low resistance recording through the skin
● Acts as a malleable extension of the electrode
○ So the movement of the electrodes is LESS LIKELY to
produce artifacts
■ Impedance
● A measure of the impediment to the flow of alternating current
○ Measured in ohms at a given frequency
● LARGER numbers mean HIGHER resistance to current flow
○ The HIGHER the impedance of the electrode, the
SMALLER the amplitude of the EEG signal
● In EEG studies should be at least 100 ohms or less and no more
than 5 kohm
○ Electrode Positioning System
■ The standardized placement of scalp electrodes for a classical EEG
recording has become common since the adoption of the:
● International 10-20 system
■ The essence of this system is the distance in percentages of the 10-20
range between Nasion - Inion and fixed points

/
 ● 21 electrodes placed at positions
○ Measured at 10% and 20% of head circumference
● The electrodes are named according to regions:
○ F = Frontal → alpha waves are strongest here (especially
on the dominant side of the subject’s brain)
○ C = Central
○ P = Parietal
○ T = Temporal
○ O = Occipital
○ A = Auricular (ear)
○ Fp = Frontopolar
○ The midline electrodes are marked with a subscript z,
which stands for zero
● Odd numbers are used as subscript for points over the left
hemisphere, and the even numbers over the right

■ We’re measuring the “alpha” waves using cap

● Measure when the subject is eyes closed vs. eyes open

/
 ■ We’re going to try to ELIMINATE artifacts:
● By characterizing their frequencies
● eg.
○ Blinking
○ Facial muscle contraction
○ Normal EEG Frequencies:

○ PROCESS:
■ Raw EEG
● Includes all noise in system
■ Band-Pass Filter
● Since we know our alpha waves are in the 8 - 13 Hz frequency
range
○ We can reject things above and below this range to
optimize and maximize the waves we collect
■ Alpha Power
● Amount of energy in the frequency band

/
 ● Take the band-pass filter signal
○ Run a Fourier transfer on it in time
● See the amplitude of the alpha wave over time

○ What to consider?
■ EEG Montages
● The placement of the electrodes
● The EEG can be monitored with either a:
1. Bipolar Montage
a. Two electrodes per one channel
b. A reference electrode for each channel
2. Referential Montage
a. A common reference electrode for all the channels
■ EEG Artifacts
● The biggest challenge with monitoring EEG is artifact
recognition and elimination
● There are:
○ Patient-Related artifacts
■ eg. Movement, sweating, ECG, eye movements
○ Technical artifacts

/
 ■ eg. 50/60Hz artifact, cable movements, electrode
paste related
○ They have to be handled differently
● These artifacts can be identified on the basis or:
○ Duration
○ Morphology
○ Rate of firing (frequency)
● There are some tools for finding the artifacts
○ eg.
■ fEMG and Impedance Measurements
● Used for indicating contaminated signal
■ By looking at different parameters on a monitor,
other interference may be found
● Particular patterns of EMG artifacts can occur in some
movement disorders:
○ Essential tremor and Parkinson's disease
■ Can produce rhythmic 4 to 6 Hz sinusoidal
waveforms
● EMG Activity
○ Common artifacts
○ eg.
1. Raising Eyebrows
a. The myogenic potentials generated in the
frontalis muscles
i. Of shorter duration than those
generated in the brain
2. Clenching Jaw
a. The myogenic potentials generated in the
temporalis muscles
i. Of shorter duration than those
generated in the brain
3. Eye Movement
a. The eyeball acts as a dipole with a positive
pole oriented anteriorly (cornea) and a
negative pole oriented posteriorly (retina)
i. When the globe rotates about its
axis, it generates a large amplitude
ii. Detectable by any of the electrodes
positioned near the eye
b. A blink causes the positive pole (the
cornea) to move closer to frontopolar FP1,
FP2 electrodes
i. Producing symmetric downward
deflections
4. Skin
a. A further difficulty arises due to properties
of certain layers of the skin

/
 b. A significant DC potential exists between
the stratum corneum and stratum
granulosum
i. Any local deformation of the skin
will alter this potential
c. The only reliable way to eliminate the
source of artifact is to create a low
resistance pathway through the layers of
skin
i. By skin cleaning (alcohol swab)
d. Also, sodium chloride (electrolyte) from
sweating reacting with metals of the
electrodes may produce a slow baseline
drift
5. Electrodes
a. Surface electrodes such as the ones used
in EEG must create an interface between
an ionic solution (the subject) and a
metallic conductor (the electrode)
i. This leads to a half-cell potential
1. Can be quite large relative
to the signal being recorded
b. To minimize this problem of polarization of
the electrode, some electrodes are coated
with silver chloride
i. But all are maintained away from
the skin through an intermediate
layer of conductive paste
c. Touching the electrodes during recording
can produce artifacts
d. An electrode which is not contacting the
skin very well acts like an antenna with
resulting 60-cycle interference
6. 60-Hz
a. The problem arises when the impedance
of one of the active electrodes becomes
significantly large between the electrodes
and the ground of the amplifier
b. In this situation, the ground becomes an
electrode that, depending on its location,
produces the 60-Hz artifact
i. Interference from high-frequency
radiation from other electronic
devices can overload EEG
amplifiers
■ The standard filtering settings for routine EEG are:
● LOW Frequency Filter: 1 Hz
● HIGH Frequency Filter: 50-70 Hz

/
● POST-LAB NOTES:
○ Overview
■ Digital Data Acquisition
■ Processing
■ EEG Waves
■ Presentation of Computer-Generated Results
○ Experiments:
1. Signal Acquisition (of a sine wave)
● By using a waveform generator
○ Box that outputs a continuous sine wave (analog)
● Continuous in TIME and RANGE of y-values
● Use an ADC (Analog/Digital Converter)
○ Transforms the continuous signal into discrete data
points (x-value, y-value)
■ At discrete TIME and RANGE of y-values

○ TWO important things to consider when digitizing data:

1. How often to record a value
a. Depends on maximum frequency within the
desired signal to record
b. Nyquist Theorem
i. The rate at which you sample signal f(t)
must be, at the very least, TWICE the
maximum frequency contained within the
signal f(t)
ii. If this requirement is not met, ALIASING
will occur...
1. Get a much lower frequency than
the actual sample
2. Non-realistic interpretation of the
data

c. Don’t get confused!!!

i. Sampling RATE = sampling FREQUENCY

/
 1. How fast you are sampling data in
time
2. Hz → points per second
ii. Sampling INTERVAL
1. 1 / sampling frequency (reciprocal)
2. Time between samples
d. PSD (Power Spectral Density)
i. Gives us information about the frequency
composition of our signal

2. Higher sampling frequencies

a. Can oversample!! (based on common
sense)
i. If points are overlapping, it’s
unnecessary
3. How to assign values that we can record
a. Depends on # of bits (determines resolution) and
sampling range
b. Amplitude Resolution
c. Computer stores information in a binary
system (0 or 1)
i. This means instead of base 10,
numbers are stored in the
computer using a base 2 system
d. Number of digits a number is represented
with in binary is the number of bits
i. The higher the number of bits, the
higher the resolution
ii. Level = 2^(# of bits)
4. Saturation
a. When choosing the ADC range, need to be
careful to prevent saturation
b. Leads to clipping of peaks
5. Applying a digital gain
a. Does not alter the frequency of the
displayed signal, only amplitude
b. Digital → named AFTER the collection of
data
i. Can take it out to restore the
original sample
6. Filtering

/
 a. Is there an IDEAL hardware filter?
i. NO!!!
ii. Ideal has an immediate, 100% cut
off…
1. All or nothing
iii. But a real filter has a gradual
frequency drop-off curve
● Measure the sine wave using a powerlab
2. Analyzing EEG Artifacts
● Introduce the things that can go wrong with EEG recordings:
1. AC (alternating current) in the power lines
a. Leave cap on bench
b. 60 Hz
c. NOT the same as thermal noise
i. Caused by random movement of electrons
ii. Power over ALL frequencies
d. Notch Filter
2. Blinking
a. High-Pass Filter
i. Prevents the low frequencies from passing
through
ii. Associated with blinking
3. Electromyographic activity
a. EMG and muscles in face
b. 50-Hz Low-Pass Filter
i. Brain waves lie between 20-50 Hz
3. Measure Alpha-Waves
● Record brain waves with an EEG signal
● Alpha waves coming from the occipital or frontal cortex
○ Alpha waves in the occipital lobe tend to be much
LARGER in amplitude
■ Much stronger when the eyes are closed (when
alpha waves are active)
○ Same seen in frontal, just less strong

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4. RESTING MEMBRANE POTENTIAL
● Purpose:
1. To measure the resting membrane potential (Em) of crayfish muscle cells
a. Using glass micropipettes
2. To study the effect of varying the extracellular potassium ion concentration on
the membrane potential
3. To study the effect of solutions containing tetraethyl ammonium chloride
(TEA) on the membrane potential
● RESTING MEMBRANE POTENTIAL
○ All living cells, under resting conditions, have a standing voltage (electrical
potential difference) between the inside and the outside of the cell (across the
plasma membrane)
■ Such that the inside of the cell is negatively charged with respect to the
outside
○ This potential is the Resting Membrane Potential
■ Its magnitude depends on the type of cell
● Usually ranges between -60 and -90 mV
■ By convention, the polarity (positive or negative) of the membrane
potential is stated in terms of:
● The SIGN of the excess charge on the INSIDE of the cell
■ Neurons and muscle cells conduct information and trigger events by
changes in this resting potential
○ The membrane potential can be accounted for by the
fact that there is:
■ A slightly greater number of negative (-)
charges than positive (+) charges inside the cell
● A slightly greater number of positive (+)
charges than negative (-) charges
outside
■ The excess negative charges inside the cell are
electrically attracted to the excess positive
charges outside the cell, and vice versa
● Thus, these excess ions collect along a
thin shell on the inner and outer surfaces of the plasma
membrane
● The bulk of the intracellular and extracellular fluid is electrically
neutral
■ The total number of positive and negative charges that have to be
separated across the membrane to account for the potential is an
insignificant fraction of the total number of charges actually in the cell
○ The resting membrane potential is determined mainly by TWO factors:
1. The differences in ion concentration of the intracellular and
extracellular fluids
2. The relative permeability of the plasma membrane to different ion
species

/
● Sodium, potassium, and chloride ions are present in the highest concentrations

○ Therefore, they generally play the most important roles in the generation of
the resting membrane potential
○ Sodium and chloride ion concentrations are lower inside the cell than outside
■ Potassium concentration is greater inside the cell
○ The concentration differences for sodium and potassium are due to the action
of a membrane ACTIVE TRANSPORT SYSTEM
■ Pumps sodium out of the cell and potassium into it
■ Na+ - K+ Pump Cycle:
1. Three Na+ ions on the inside of the cell membrane bind to the
pump protein (carrier molecule)
a. The pump protein is phosphorylated by ATP
2. The 3 Na+ ions are released to the outside of the cell membrane
3. The outside 2 K+ binds to the pump protein
a. 2 K+ is released to the inside of the cell
4. The pump protein releases the phosphate
a. Returns to its original conformation
● How do CONCENTRATION DIFFERENCES create membrane potentials?
○ Let us assume that the membrane is permeable ONLY to potassium
■ Not permeable to chloride and sodium
○ Therefore, potassium can diffuse through the membrane but chloride and
sodium cannot
○ SCENARIO:
■ Initially there is NO potential difference across the
membrane
■ Because the two solutions are electrically neutral
● eg.
○ They contain equal numbers of
positive and negative ions
○ The positive ions are different
(sodium vs. potassium) on the two
sides
■ But, the numbers are the same
○ Each is balanced by an equal number of chloride ions
■ Because the membrane is permeable to
potassium ions, they will flow down their
concentration gradient
● eg. towards the outside of the cell
■ There is ALSO a concentration gradient
favouring sodium diffusion in the opposite
direction

/
 ● BUT, the membrane is not permeable to sodium
■ Accordingly, after a few potassium (positive) ions have moved out of
the cell, the cell will have an EXCESS of negative (-) charge
● Whereas the outside solution will have an EXCESS of positive
(+) charge
■ A potential difference will now exist across the membrane
● POTENTIAL DIFFERENCE
○ Influences the movement of potassium ions
■ Being positive (+), they are:
● ATTRACTED by the negative (-) charge on the intracellular side
of the membrane
● REPULSED by the positive (+) charge on the extracellular side of
the membrane
○ As long as the force due to the concentration gradient
driving potassium ions OUTSIDE the cell is greater than the
electrical force driving it INSIDE:
■ There will be net OUTSIDE movement of potassium
ions
○ The cell will become more and more negative (-) UNTIL:
■ The electric force opposing the exit of potassium
ions outside of the cell equals the force due to the
concentration gradient favouring its exit
○ EQUILIBRIUM Potential
■ The membrane potential at which the electrical
force is equal in magnitude but opposite in direction to the
concentration force for an ion
■ At the equilibrium potential there is NO net movement
of the ion
● Because the opposing forces acting on it are
EXACTLY BALANCED
■ The value of the equilibrium potential for any ion
depends upon the concentration gradient for that ion
across the membrane
● If the concentrations on the two sides were
equal:
○ The force of the concentration
gradient would be zero
○ The equilibrium potential would also
be zero
● The LARGER the concentration gradient, the
LARGER the equilibrium potential
■ The equilibrium potential for any ion can be
calculated using the so called Nernst Equation
● EXPERIMENTS
○ Hypothesis:
■ The muscle membrane at rest is exclusively permeable to potassium
○ If this hypothesis is valid:

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 ■ The resting membrane potential should be the SAME as the equilibrium
potential for potassium:

■ Therefore it should be possible to predict the changes in resting

membrane potential for various [K+]o using simply the Nernst equation
which for potassium ions is:

● Variables/Constants:

● At room temperature:

○ Thus a graph of Em to log10 [K+ ]o should be a straight line with a slope of 58.
■ eg.
● A 10-fold change in [K+ ], Em should change by 58 mV
● A 100-fold change in [K+]o, Ek will change by 116 mV (58 + 58).
○ For physiological conditions, we will assume that [K+]i = 139 mM
■ In the graph below, Ek is plotted, assuming that a [K+]i of 139mM
remains the same as [K+]o changes

● In the graph, note the general trend of the Ek:

○ With increasing [K+]o, Ek is reduced (becomes less
negative)
● If Em = Ek, then the graph for Em should appear similar to that
for Ek as given above
■ At physiological levels of [K+ ], the measured membrane potential is
usually LESS negative than the potassium equilibrium potential (EK)

/
 ● Mainly because the Na+ permeability although small, is NOT
zero at rest
○ Thus, we will conclude that ALTHOUGH Em is close to Ek,
it is NOT exactly equal
■ Especially at low [K+]o
■ Due to the influence of Na+
● LEGEND:
○ Circles → the measured membrane potentials at various
[K+ ]o
○ Straight Line → the relationship between the potassium
equilibrium potential and [K+ ]o
■ Calculated from the Nernst equation
● The influence of other ions is best determined using the Goldman Equation

○ Similar in form to the Nernst Equation

○ BUT, incorporates permeability to Na and Cl
■ In fact, the inclusion of chlorine DOES NOT appreciably affect the
solution of the equation
● RMP: Recording Circuit
○ The circuit used for measuring the membrane
potential
○ Composition:
■ A recording glass electrode is lowered into a
muscle cell
■ Electrode
● A conductor through which electricity
enters or leaves an object, substance,
or region
● Made of materials which can
participate in a reversible reaction
with one of the ions in the solution or electrolyte

/
 ○ This permits the CONVERSION of ionic current in solution
into electron current in wires
● The most frequently used electrode material in
electrophysiology is a silver (Ag) wire coated with
silver-chloride (AgCl)
○ The following reaction takes place in a solution filled
glass micropipette: Cl- + Ag <--> AgCl + e-
■ A glass micropipette is heated and pulled
to a fine tip
● Very small tip diameters can
minimize the electrolyte from
entering the cell and changing its
normal anion and cation content
○ However, this is done at the
expense of:
■ Noise
■ Diminishing current
passing ability
■ Limiting recording
bandwidth
● It is then filled with the electrolyte
solution
○ Provides the necessary fluid bridge between the cell and
the electrode
● The composition of the electrolyte depends on the TYPE of
measurement made
● The concentration of the electrolyte is also important
○ HIGH electrolyte concentrations:
■ Reduces the electrode resistance
■ Lowers voltage noise
■ Provides a wider recording bandwidth.
● Filled with:
○ 3 M KCl
○ A fine chlorided silver wire (inserted inside)
■ The voltage measured is then sent to a high impedance preamplifier
■ Then, sent to the recording system
● The microelectrode holder is designed to provide an electrical
coupling between the fluid-filled glass pipette and the high input
impedance preamplifier
○ The wire fits inside the shaft
of the electrode above
● The glass pipette is secured inside
the holder by adjusting the Plexiglas
screw
○ The linear relation between potential difference
and current flow, as given by Ohm's law applies to
aqueous ionic solutions
■ eg. cytoplasm

/
 ■ However, certain complications arise due to the following problems:
● TIP POTENTIAL
○ The Ag/AgCl electrode performs well only in solutions
containing Cl- (see the equation above)
○ Since current must flow in a complete circuit:
■ TWO chlorided silver electrodes are needed
○ If the electrodes are immersed in two different
concentrations of chloride solutions, there will be a
difference in the half-cell potentials at the two
electrodes
■ eg. when one electrode is in the bathing fluid of
the muscle and the other inside the micropipette
○ This tip potential can be subtracted electronically or
compensated by adjusting the voltage offset

○ The pre-amplifier used in the recording of membrane

potential:
■ It has connections to the bath electrode, and the
micro-electrode
■ The output goes to the Powerlab recording
system
■ The position knob adjusts the baseline to zero,
when the tip potential is recorded
○ Note that, in the circuit used, the tip potential can be
easily compensated by displacing the “position” knob on
the pre-amplifier
■ So that the computerized recording system
returns the baseline trace as zero
■ The membrane potential can then be read as the
new potential difference from the zero trace
○ Pre-Amplifier
■ The amplifier used is a special DC, high resistance (>10^10 ohms) input
unit
■ The main function of this amplifier is to act as a resistance matching
device
● Between the microelectrode (high resistance) and the recording
system (low resistance)
■ The reason why this high input impedance amplifier is needed is
explained below:

/
 ● Cases:
○ WITHOUT the high resistance amplifier
■ During the recording, current will flow through
BOTH the high resistance of the recording
electrode (Re) and the low resistance of the
recording system (Ro)
● Since these two resistances are in series,
the total resistance RT = Re + Ro.
■ Because the sum of the two resistances is quite
low, the current is VERY HIGH
● This is undesirable as it will alter the ionic
environment of the cell.
■ During the flow of this current, a large fraction of
the total Em will appear as a voltage drop across
the microelectrode
● Only a small fraction of the total voltage
drop will occur across the recording
system:
■ Since Vo is our estimate of Em, we naturally want
Vo to equal Em as closely as possible
● In the case above, Vo will be only a small
fraction (< 1/10) of the actual Em
● This is obviously undesirable
■ In addition, most of the voltage drop (99.9%) will
occur across the amplifier, and not across the
microelectrode
○ WITH the high resistance amplifier
■ The current flow will be much reduced
● In addition, most of the voltage drop will
now occur across the amplifier
● Not across the microelectrode
■ Hence, the recorded potential will be almost
identical with the actual Em
● Therefore using the high resistance
amplifier has allowed us to:
1. Accurately measure the resting
membrane potential
2. Significantly reduce the current
flowing in the circuit
■ The sensitivity of the Em estimate (V) to changes
in R (if the electrode is slightly plugged or broken)
will be greatly reduced
● PROCEDURE
1. Crayfish Dissection
2. Analysis of Data
a. Mean Membrane Potential

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 i. 5 deflections of approximately equal amplitude
1. The membrane potential for each penetration is
calculated by subtracting the Tip Potential from the Tip +
Membrane Potential
ii. From the determined membrane potentials, the mean
membrane potential will be calculated
1. There will be some variation in each solution data set,
and this will be reflected in the standard deviation
iii. Step-like data similar to the above will be obtained for all 9
solutions
1. 6 solutions without TEA
2. 3 with TEA
iv. For solutions with HIGHER extracellular concentrations of [K+]o,
the step-size will be smaller
1. Correspondingly, the membrane potential will be smaller
(less negative)
b. No TEA (Tetraethyl Ammonium Chloride)
i. Normal Ringer’s
ii. If the experiment was performed correctly, the graph might look
somewhat like the one to the left
1. The slope of the first four points is LESS than that of the
last four points
a. This is because at lower [K+]o, Na+ ions
contribute MORE to the membrane potential
i. Remember that although the membrane is
permeable mostly to K+, it is slightly
permeable to Na+
ii. At low extracellular concentrations of K+,
the [Na+] ions have more of a tendency to
move into the cell due to a high driving
force acting on the Na ions
1. Thus, making the membrane
potential less negative
2. As a result, the slope is more gentle
b. At high [K+]o however, the Na+ ions play less of a
role
i. The driving force pushing Na into the cell
is not as great
1. Thus, Em is close to Ek
ii. The slope of the line approaches 58
iii. We would expect a slope of 58 if Em = Ek
iii. SIDE NOTE:

/
 1. Driving Force
a. A measure of how far those ions are from their
equilibrium potential (ENa)
i. Assuming ENa is somewhere around +60
mV, you should be able to visualize looking
at the graph above, how much greater the
driving force will be at low rather than
high [K+]o
b. A form of potential energy
c. With TEA
i. The data is plotted and a line is fitted through the points
ii. If the experiment was performed correctly, the graph might look
somewhat like the one to the right below

1. The slope of the points is considerably less than what we

obtained without TEA above
iii. TEA is a compound that BLOCKS potassium channels
1. If potassium permeability has been reduced, we expect
the membrane potential to move more towards sodium's
equilibrium potential (Ena)
3. Calculations
a. Mean
i. Average of the numbers
b. Variance
i. Subtract the mean from each number
1. Square the result
ii. Find the average of those squared differences
c. Standard Deviation
i. Square root of the variance
d. Standard Error of the Mean
e. T-Test
i. Performed to determine whether there is a significant difference
between the mean values of Em for the two extreme solutions:
1. [K+]o = 0.1 mM
2. AND [K+]o = 60 mM
ii. If we set a level of significance at 5%, then we mean that:

/
 1. There is a 5% probability that the difference in means
can be attributed to chance
2. Alternatively, a 95% probability that the difference is
NOT attributed to chance
iii. We must first calculate the degrees of freedom of the system
1. The degrees of freedom are given by: 2n – 2
a. n = number of measurements in each group
i. Assumed to be the same
b. So for 10 values: 20 - 2 = 18
iv. We next look up, in a probability table, for 18 degrees of
freedom at a significance level of 5%
1. T value which will determine whether we should accept
or reject the null hypothesis
2. In this case:
a. Reject the hypothesis if the calculated t is > 2.10
b. Conclude that there is a significant difference in
the two means
i. Would mean that there is a relationship
between the external potassium
concentration and the resting membrane
potential
● POST-LAB NOTES:
○ THREE Terms:
1. Resting Membrane Potential
a. The electrical potential difference across the plasma membrane
i. Difference between the intracellular matrix by the cell
membrane and the extracellular matrix by the cell
membrane
b. Governed by the:
i. Concentration gradient of ions
ii. Permeability of the membrane to these ions
2. Equilibrium Potential (for an ion)
a. The membrane potential at which the force from the electrical
gradient is EQUAL in magnitude but OPPOSITE in direction to
the force of the concentration gradient
3. Driving Force (for an ion)
a. The propensity of an ion type to cross and move through the
plasma membrane
b. Essentially, a measure of how far those ions are from their
equilibrium potential (Eion)
○ Cell
■ THREE main Ions
1. K+
a. Higher concentration INSIDE the cell
2. Na+
a. Higher concentration OUTSIDE the cell
3. Cl-
a. Higher concentration OUTSIDE the cell

/
○ Nernst Equation
■ Measures the equilibrium potential for a specific ion
■ eg. K+
● If the membrane was only permeable to potassium, then its
equilibrium potential would be this value
○ Based on the concentration values that you fill in,
respectively

■ Plotted
● Slope of Ek = 58 (at room temperature)

● If our hypothesis that Em = Ek is true, our data points would all

fall along this line
○ BUT, this is not true…
■ At low extracellular [K+], Em is NOT exactly Ek
● Slope is steeper
■ As you increase the extracellular [K+], the slope
would have increased
● Approached 58

/
○ LAB QUESTION SOLUTIONS:
1.
a. Degrees of freedom = 2n-2 (n is # of samples, 5 cells)
b. P-value
i. Represents the probability that the difference in the two
concentrations of K+ (high and low) is due to random
chance
ii. %
1. P-value of 5 % is a P-value of 0.05 (not 5)
iii. eg. p = 5%
1. If we did this experiment 100 times, 5 of those
times we would get the correct difference
a. But it’s by chance
iv. 100% - (P-value)% = how confident you are that the
observed difference is reliable
1. The lower the p-value, the more confident you
can be in your results
v. eg. 100 % - 5 % = 95 % confident that your observed
difference is due to your experimental hypothesis
c. To test if it’s statistically significant:
i. Compare t-values
ii. If your experimental t-value > the t-value for your
P-value (based on your degrees of freedom)
1. YES
2. The difference is statistically significant
d. Why estimating p and t-values is more reliable than simply
reporting the mean-values and their differences?
i. The t-value considers the VARIABILITY of the data (i.e.
standard errors of the means) too
ii. Just calculating the difference between two means does
not
2.
a. Na+ shifts the equilibrium potential more at lower [K+]
b. Not true difference since the Nernst equation does not take into
account all the ions
i. Should use Goldman's equation

/
3.
a. N/A
4.
a. log(10) = x1
b. log(100) = x2

c. A change
i. Can be + or -
ii. Based on how you are changing the extracellular
concentration
5.
a. Lower [K+] not as steep
i. Driving force is Na+
6.
a. Once you add TEA, the slope becomes less steep at higher [K+]
i. Since TEA blocks K+ channels
ii. Slope would be similar to lower [K+]

b. Membrane potential would have been greater (more +)

i. Closer to Na+ equilibrium potential
7.

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 a. Slope is less steep
8.
a. A drop sometimes falls out from the tip of the microelectrode
i. If it was filled with NaCl and the drip had occurred inside
the cell
1. Would change our membrane potential
significantly
a. Since there isn’t much Na+ to begin with…
2. eg. dropping water in a glass
ii. Since the intracellular [K+] is already so high, adding a
drop will not affect the results greatly
1. Eg. dropping water into the ocean

9.
a. To reduce the current flowing through
i. You don’t want a high current flowing through our
recording electrode
ii. Might alter the ionic concentrations within our cell
b. To accurately measure the resting membrane potential
i. Amplifier creates a resistance GREATER than the
resistance of the electrode
1. Allows the voltage drop to occur over the
recording system
ii. Could capture close to 100% of the actual membrane
potential
c. To eliminate the sensitivity of the recording to changes in the
electrode resistance
i. The tip was easy to break or be plugged
1. Would change the resistance of the electrode
ii. But you want to keep the recordings the same from one
recording to another
1. High resistance prevents these from changing
your data significantly

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/
5. ACTION POTENTIAL
● Purpose:
○ Study some basic features of nerve excitation and impulse conduction
○ To examine the behavioral response of the worm to TOUCH:
■ These neurons are important for touch-mediated escape responses
■ We can activate the fibers with touch or with electrical stimulation of
the nerve
● Facilitates movement away from the stimulus
■ Touching the HEAD activates the Medial Giant Fiber (MGF)
● Touching the TAIL activates the two Lateral Giant Fibers (LGF)
○ To record extracellular action potentials (APs) from the ventral nerve cord of
the earthworm, Lumbricus Terrestris
1. To study the characteristics of the earthworm AP
a. Latency/Delay
b. Time Course
2. To measure the nerve conduction velocity:
a. How fast an AP moves along an axon
i. Varies on the axon diameter and whether or not it is
myelinated
3. To construct a strength/duration curve of the nerve
● Content:
○ NERVE FIBERS
■ An extension of a neuron that consists of an axon and, in some cases, a
myelin sheath
● TRANSFERS INFORMATION, in the form of action potentials,
from one part of the nervous system to another
■ Regardless of the type or size of the fibers, they ALL conduct
non-decremental (ALL-OR-NONE) action potentials
■ However, their Conduction Velocity varies depending on their:
1. Size (diameter)
a. LARGER → more rapid conduction velocity
i. Larger diameter axons are also easier to
stimulate!!
2. Myelinated vs. Unmyelinated
a. MYELINATED → more rapid conduction velocity
i. “Saltatory Conduction”
1. Potential will only need to change from
node to node
2. Allows the AP to move much more rapidly
down the length of the axon
ii. The relationship is approximately:
1. Velocity (m/s) = Diameter (micrometers) x
2.5
b. UNMYELINATED

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 i. Continuous depolarization of the membrane
potential along the axon

● By definition, the conduction velocity of the AP is given by:

○ d/t
■ d = the conduction distance
■ t = the conduction time
○ Reported in (m/s)
■ It is more easily recorded as millimeters per
millisecond, which yields the same result
● You want to measure conduction velocity in a neuron that does
not continuously fire a lot of action potentials…
○ eg. why giant axons are so useful!!

○ Otherwise, it would be difficult to determine which AP is

being fired

■ VERTEBRATE
● Nerves consist of thousands of axons

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 ○ eg. the vagus nerve in man consists of over 100,000
fibers
● The cell bodies of these axons are located either in the:
1. CNS
a. For motor fibers
2. PNS
a. For sensory fibers
i. eg. Dorsal root ganglia
● Fibers/axons may be:
1. Large and Myelinated
a. 15 - 25 µm
2. OR Small and Unmyelinated
a. 0.2 µm
● In general, small (less than 25µm), myelinated and unmyelinated
fibers are a feature of vertebrates
○ However, diameters can vary EXTENSIVELY!!
● eg.
○ Touch fibers have much HIGHER conduction velocities
than pain fibers
○ Why, when you stub your toe, you feel the pressure
before you feel it hurting

■ INVERTEBRATES
● Invertebrate nerves can consist of a SINGLE, GIANT AXON
○ For rapid conduction of action potentials
○ Important for fast escape responses
● In general, large (up to 500 µm or larger), unmyelinated fibers
are a feature of invertebrates
○ Studying these giant invertebrate nerve fibers has been
important to our understanding of:
1. Action Potentials
2. Ion Channels
a. Control changes in membrane potential
● eg. Hodgkin and Huxley (1952)
○ Studied the giant axon of the SQUID
■ Up to 1 mm in diameter
● For comparison, the largest human axons
are 0.1 mm in diameter

/
 ○ Discovery:
■ At rest, a neuron is more (-) charged inside than
out
■ During the AP, sodium channels in the membrane
open
● Sodium ions rush in
● Results in membrane depolarization
■ When the membrane potential reaches a
threshold, it triggers the opening of neighboring
sodium channels
● This positive depolarization continues and
propagates the action potential down the
length of the nerve fiber
● eg. EARTHWORM
○ Worms belong to the class of invertebrates known as
annelids
○ In our experiments we will use the earthworm,
Lumbricus Terrestris

■ The dorsal (top) side of the worm is DARKER than

the ventral (bottom)
○ Majority of the earthworm body is made up of its GUT
(surrounded by muscles)
■ The remainder of the nerve cord is largely
neuropil (dense network of intertwining fibers):
● Where synaptic connections are made

○ “Nerve Cord” is near the VENTRAL (bottom) surface of

the worm

/
■ Contains three giant axons that run across the
length of its body
● Found near the dorsal (top) surface of the
nerve cord
■ Each axon is formed from many individual
neurons
● Axons fuse into a single, functional unit
● BUT, the cell bodies remain separate
1. “Medial Giant” fiber (MGF)
a. ONE
b. Connected to the HEAD of the worm
i. Head region is closer to the
clitellum (swelling)
c. Head → Tail
i. Transmits sensory information
about the anterior of the worm
d. LARGER
i. Conducts more rapidly
ii. Diameter → 0.07 mm
2. “Lateral Giant” fibers (LGF)
a. Connected to the TAIL of the worm
b. Tail → Head
i. Transmit information from the
posterior end of the worm
c. SMALLER
i. Diameter → ~0.05 mm
d. TWO
i. Interconnected by electrical
synapses at many points along
their length
1. Normally fire together
ii. Together, they contribute to only
ONE spike in our extracellular
recordings
■ These nerves conduct in OPPOSITE directions
● However, when action potentials are
electrically stimulated, the spikes
propagate away from the stimulating
electrodes in BOTH directions
■ These nerves are necessary for the “escape
withdrawal reflex”
● The organism detects and processes
information about a tactile stimulus → tap
to the head or tail
● Converts the tactile stimulus into an
electrical signal → neuronal APs
○ APs are then propagated
throughout the animal’s body

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 ● Causes succinct muscular contractions
○ Although earthworm “giant fibers” are much smaller than
the squid giant axon (1 mm diameter),
■ Earthworms have the advantage of being EASY to
obtain and to keep in the lab
○ ACTION POTENTIALS
■ A transient (short), depolarizing SPIKE
● Brief electrical impulses
■ Propagate information from one region of the nervous system to
another
● Stimulation usually comes from the level of the DENDRITES

● Moves down the axon

○ Usually start at the initial segment of an axon
○ Propagates down the length of the axon to the
presynaptic terminals
■ This signals the next neuron in the chain
■ There’s nothing that causes the UNIDIRECTIONAL movement of the AP
● It’s just by the virtue of the depolarization of the membrane…
■ So, if we had electrically stimulated the nerve in the middle of its axon:
● The AP would propagate in BOTH directions

■ BIPHASIC Waveform (+ and - regions):

● Resting Membrane Potential → -70 mV

1. When the nerve receives a stimulus that causes DEpolarization

a. Voltage-gated Na+ channels OPEN
i. Na+ (+) flows INTO the cell

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 b. If the depolarization is small, it will result in transient
increases in membrane potential
i. These are not strong enough to activate the AP →
failed initiations
c. THRESHOLD
i. The MINIMUM stimulation voltage needed to elicit
an action potential
ii. Depends on the:
1. PROXIMITY of the stimulating electrodes
to the axon target
2. The SIZE of the axon
a. Larger diameter axons have larger
ionic currents flowing around them
d. AP is an “all-or-none” event
i. As long as it PASSES the threshold, it will produce
an AP
ii. At the AP peak, the membrane potential
approaches Loading… → +40 mV
e. ABOVE a threshold, the AP size does NOT change with
increasing stimulus intensity
2. Followed by REpolarization
a. Voltage-gated Na+ channels CLOSE
b. Voltage-gated K+ channels OPEN
i. K+ (+) flows OUT of the cell
c. ABSOLUTE Refractory Period
i. Membrane is COMPLETELY unexcitable
1. A brief period in which the Na+ are
recovering from inactivation
ii. Does not matter how large of a stimulus is
provided to the neuron…
1. It cannot be activated
d. RELATIVE Refractory Period
i. After the absolute refractory period, there are
enough Na+ channels in the closed (active) state
to respond to depolarization
1. However, K+ channels that opened in
response to repolarization close much
slower
2. Stays open for much longer...
ii. Results in HYPERpolarization
1. Brief OVERSHOOT of the membrane
resting potential
2. Resting membrane potential goes BELOW
the resting membrane potential
a. A more (-) value
iii. Thus, the membrane is at a higher threshold
(farther than the normal threshold)

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 a. But it would require a LARGER
stimulation
2. Since it is LESS excitable
3. Eventually relaxes back to its resting state

■ Why record AP’s?

● Basic Science:
○ To find out HOW nerves operate
○ Since AP’s are the way that nerves transmit information
● Clinical:
○ To DETECT nerve or muscle malfunction in patients with
neurological disorders
○ eg. Nerve Conduction Velocity Tests
■ Used for diagnosis of various neuropathies
● Especially demyelinating conditions:
○ Results in reduced or non-existent
conduction velocities
● To determine whether muscles are
functioning properly
○ In response to stimuli sent via their
connecting nerves
■ Process:
1. Place two electrodes (intra or
extracellularly) on the patient
a. Need to know the DISTANCE
between these two electrodes
2. Apply a pulse to one electrode
a. Measure the time the pulse takes to
reach the second electrode
(conduction velocity)
3. Can check if the nerve conduction is
normal AND if the muscle is responding
a. So, you can differentiate whether
there is a problem at the level of
the nerve or the muscle synapse
itself…
■ Neuronal APs are NOT the only way for cells to communicate
● Diffusion of ions/hormones!!!

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 ■ In single-celled animals, simple diffusion is sufficient...
● But, too slow for use in LARGER animals:
○ Since diffusion is proportional to the square of the
distance
■ Takes place over longer time frames and
distances
○ NOT efficient for rapid communication
● eg. Paramecium
○ Lives in pond water
○ Covered in flagella
■ Beating of this flagella propel the organism
through its environment
○ Behaviour is dependent on the concentration of internal
Ca2+
■ If it bumps into something, it opens Ca2+
channels at one end of the animal
● Leads to depolarization
● Increases the Ca2+ INSIDE
○ This rise of Ca2+ causes a reversal in the feet direction
■ eg. forward to backward

○ RECORDINGS
■ INTRAcellular
● Allows for very accurate assessment of the electrical activity of
a single cell
○ Measures the ACTUAL change in membrane potential
● Best used for large invertebrate cells
○ Where you don’t have to worry about damaging the cell
by puncturing it
● It is very difficult to do in vertebrate nerve fibers
○ Can cause considerable damage to the membrane
around the electrode tip
● Process:
1. Insert a glass pipette into a cell
2. Record the potential changes
a. With respect to an extracellular reference
electrode
● Biphasic waveform (+ and - deflections):

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 ○ A large (+) depolarization
■ Due to a transient increase in Na+ permeability
○ Followed by a smaller and slower (-) hyperpolarization
■ Caused by a persistent increase in K+ permeability
● Peak Amplitude → ~100 mV
■ EXTRAcellular

● For SMALLER neurons or nerves

● Best suited for measuring:
1. If an AP has occurred
a. Not necessarily looking at the waveform itself
b. OR the change in membrane potential
2. The ACTIVITY in a population of cells
● Process:
○ Place stainless steel pins (recording electrodes) near the
excitable cell
■ Place reference electrode at some location in the
extracellular fluid
■ In lab:
● Place in the worm
● NOT near or in the cell of interest
○ Records potential changes at the membrane SURFACE
■ Measures the local circuit currents flowing around
an axon as the action potential propagates
● The change of potential NEAR the
membrane surface
■ NOT measuring the ACTUAL changes in
membrane potential
○ Peak Amplitude → < 1 mV
■ Need to be amplified because so small!!
● Advantages:
1. Relatively easy to do
2. Does NOT damage the cell membrane
a. We don’t have to worry about killing the cell that
we are recording from
● Disadvantages:
1. AP size will be much smaller (reduced)
a. Since we are measuring extracellularly
b. Microvolts vs. millivolts

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 2. AP waveform we see depends on the exact geometry of
electrodes relative to the position of the cell (the contact)
● Biphasic waveform (+ and - deflections):
○ (-) Phase
■ Dependent on WHERE the two pin electrodes are
placed with respect to the travelling AP
● The “recording technique”
■ Not like intracellular recordings, where it is based
on the after-hyperpolarization....

○ Because the potentials are so small, it requires Differential

Recording
■ Measuring the difference between the two recording
electrodes (+ and -) relative to a ground electrode
■ Procedure:
● Metal pin recording electrodes placed near the
nerve
● TWO PAIRS
○ One each for CH3 and CH4
○ Each pin in the pair (+ and -) is connected
to one input of the differential amplifier
■ Ideally, make sure the difference
between the + and - is the SAME in
both pairs
● Before the stimulus is delivered, both pins should
be measuring basically the SAME voltage:
○ So, because the amplifier takes the
difference of the two inputs, there should
be no deflection recorded
● The situation changes as the AP travels along the
nerve...
○ The shape of the AP will depend on the:
1. Relationship between the
inter-electrode DISTANCE

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 2. LENGTH of the axon segments
depolarized by the action potentials
3. Conduction VELOCITIES of the
axons
● When the AP has reached the first recording
electrode (Ch3, proximal):
○ All the potential is at the first electrode...
○ The proximal electrode becomes
transiently (-) to the distal electrode
○ The potential difference between the two
is detected
■ The trace is displayed as an upward
deflection on the screen
● As the AP progresses between the two recording
electrodes:
○ Before the AP reaches the second
electrode...
○ The recorded potential returns to the
baseline
■ NO voltage difference between the
two recording electrodes
■ A short segment of 0 deflection
■ This occurs IF the electrodes were
sufficiently FAR apart
○ However, if the electrodes are NOT
separated by such a large distance…
■ The AP will not have completely
passed the first electrode before
reaching the second
■ The two phases will NOT be of
equal amplitude
■ Adding the two opposite signed
deflections will REDUCE the
amplitude of the negative phase
■ Would DECREASE the apparent
width of both
● As the AP passes the second electrode (Ch3,
distal):
○ All the potential is at the second
electrode...
○ A deflection of the same size but opposite
sign will be recorded
■ The sign is (-) because of the way
the amplifier compares the two
inputs
● Once it passes the second electrode, it returns to
its baseline (flat line) → 0 deflection
○ STIMULATION

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 Electrical
■
● At head:
○ We could be stimulating either the medial giant fiber OR
the lateral giant fiber…
○ We don’t know which one we’re activating
● Same is true from the opposite end (tail)
■ Touch
● At head:
○ Only activate the medial giant fiber
● Same is true from the opposite end (tail)
○ EXPERIMENT
■ Overview:

i. Behavioural Responses (touch):

ii. Action Potential:

1. The second biphasic waveform
a. The Stimulus Artifact appears first
2. It is important not to confuse the two...
a. Stimulus Artifact:
i. Initially with a low initial stimulus amplitude, no
AP will be visible
1. But, you will see a brief, biphasic
deflection near the beginning of the
display
ii. Results from virtually instantaneous, passive
current spread from stimulating electrodes to
recording electrodes
b. Threshold Stimulus Voltage

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 i. Determined by raising the stimulus voltage to find
the voltage at which the AP is just discernible
1. Adjust the duration and the volts on the
stimulator until we see an AP firing

ii. This is the actual nerve Action Potential

3. Because this an “all-or-none” event, increasing the stimulus will
NOT increase the size of the AP
iii. Extracellular Recording Configuration:

iv. Characteristics of AP (touch vs. electrical):

1. Taken from channel 3 (the first AP)
2. Latency/Delay
a. ONSET of the AP
i. The time from the onset of the stimulus artifact
to the onset of the AP
b. PEAK of the AP
i. The time from the onset of the stimulus artifact
to the peak of the AP.
3. Threshold
a. Threshold Stimulus Voltage
i. Determined by raising and lowering the stimulus
voltage a little
1. To find the voltage at which the AP is just
discernible
b. Maximal Stimulus Voltage
i. The point at which a further increase in stimulus
voltage produces no further increase in the AP
amplitude.
4. Shape
a. Why does the AP increase in size and duration with
increasing stimulus strength?
i. The AP is the algebraic sum of all individual APs

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 5. Peak Amplitude
a. The voltage value of the peak of the AP response
6. Duration
a. The time from the beginning of the (+) phase to the end
of the (-) phase of the AP

v. Conduction Velocity (electrical vs. touch)

1. Measure the time between the peak of the AP on Ch3 and the
peak of the AP on Ch4
a. Measure the distance between the two proximal
recording electrodes

2. Depolarization of an excitable membrane requires a flow of

electrical charge across the membrane
a. Because of the DOMINANT electrical capacitance of the
membrane:

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 i. The relevant parameter for effective membrane
depolarization is the total amount of CHARGE
transferred across the membrane
3. To OVERCOME the threshold...
a. For a short duration stimulus generating a steady
trans-membrane current, the charge transferred (Q) is
proportional to the product of:
i. Current (I) → strength
1. Can use voltage (V) or current (I) as the
measure of stimulus strength
ii. AND Time (T) → duration
b. Q = I x T or I = Q / T
i. According to this equation (a hyperbola):
1. If the neural membrane was an IDEAL
capacitor:
a. The effective stimulus intensity
should decline asymptotically
towards the baseline
i. As stimulus duration is
increased...
b. Thus, even a very weak stimulus
should be effective if its duration is
prolonged
ii. This suggests that a graph of threshold stimulus
STRENGTH vs. stimulus DURATION should show:
1. A decline to near zero as stimulus duration
is increased
a. In other words, the stimulus
strength required to reach threshold
should DECREASE during more
prolonged stimulation
vi. Strength-Duration Curve (stimulus)
1. The ease in which a membrane can be
stimulated depends on:
a. STRENGTH of stimulus (applied current)
b. DURATION of stimulus
2. Variables are inversely related
a. eg. as the strength of the applied
current increases, the time required to
stimulate the membrane decreases
3. These curves can tell us properties of a neuron:
a. Rheobase (V)
i. LOWEST intensity stimulus with indefinite pulse
duration that stimulates an AP
1. Where the curve asymptotes
ii. The rheobase never actually asymptotes at 0...
since in real neurons, there are:
1. Leak Currents across the cell membrane

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 a. Under resting conditions, an
outward current opposes the
inward current
i. Outward → K ions via the
K+ leak channels
ii. Inward → Na+ current
induced by stimulation
2. Imperfect Recording Conditions
iii. When stimulus strength is BELOW the rheobase,
stimulation is ineffective
1. EVEN when stimulus duration is very
long...
2. Since the (outward) leak current EXCEEDS
the (inward) stimulating current
a. There can be NO excitation
b. Chronaxie (ms)
i. MINIMUM stimulus time required for an electric
current to double the strength of the rheobase
1. 2 x rheobase
ii. We need to determine the TIME at which this
occurs
4. blue line → amount of charge (Q)
a. For a HIGH amplitude stimulus, the time required to
stimulate the membrane is SHORT
b. For a LOW amplitude stimulus, the time required to
stimulate the membrane is LONG

● FINAL RESULTS:

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● POST-LAB NOTES:
○ Objectives of Lab:
1. Understand the compound action potential (CAP)
2. Understand differential recording
3. Make inferences on the nerves
a. Given the differences between touch and electrical stimulation
4. Construct a strength/duration curve of the nerve to get rheobase and
chronaxie
○ Intracellular vs. Extracellular Recording:
■ Intracellular
● Movement of ions
● Accurate assessment of electrical activity
● But, can damage membrane
■ Extracellular
● Detecting the “electric fields” produced by the movement of ions
● The size of any one action potential will be reduced
○ Voltage will also decrease
● Shape of waveform depends on the exact geometry of its
contact with the electrode
○ COMPOUND Action Potential (CAP)
■ Sum of many action potentials
● Perhaps from different diameter fibers
■ Summed AP’s from the medial and lateral fibers (cells) → three nerves
● Form the ventral nerve cord
■ Relationship to ion flow
● Intracellularly
○ AP causes cell to be more positive (influx of Na+, efflux
of K+)
○ Depolarized
● Extracellularly
○ Result in a wave of negative charge
■ The outside of the cell becomes transiently more
negative
○ Mirror image of what is happening intracellularly…
● The AP goes in both directions
○ We record one based on how the electrodes are placed
○ Worm Anatomy
■ They breathe through their skin
● As long as it stays wet!
■ Citellum
● Bump
● Stores the eggs of the worm
● Species is hermaphroditic
○ Both male and female sex organs
■ Lateral giant fibers are FUSED
● Think of them as a single cell
■ No myelin
● Since worms are invertebrates

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 ● There are glial cells that support the nerves
○ LAB QUESTIONS:
1. Escape Withdrawal Reflex
a. Response of worm to a gentle touch to anterior/posterior
b. Shortening away from stimulus
i. Muscle contraction
ii. Reflex to escape from predators
c. Impaired after anesthesia
2. Electrode Layout

a. Stimulating Electrodes (placed on both ends of worm)

i. Cathode (-) more towards center
ii. Anode (+) further from center
iii. Why is the cathode (-) more towards the center?

1. Cathode (-) causes the AP

a. Deposits (-) charges on the surface of the nerve
i. (+) charges are attracted to it on the inside
of the nerve
b. The (+) environment causes depolarization
i. Potassium voltage-gated channels open
ii. AP occurs
2. Anode (+) attracts (-) charges
a. Attacks (-) charges from the cathode
i. “Vacuums” them up
b. Causes an anodal block

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 i. The nerve underneath the anode is
hyperpolarized
1. More (+)
ii. (-) environment inside the nerve
1. More difficult to drive an AP
2. Threshold goes up
3. Why the anode has to be AWAY from the direction we
wish to measure the AP
a. If it was flipped, the threshold would INCREASE
b. Making it more difficult for the AP to propagate, if
it wasn’t completely blocked
b. Recording Electrodes (two channels)
i. Cathode (-) to the left of the anode (+)
c. Ground
i. Between the cathode (-) of a stimulating electrode and the first
channel that you record from
3. Conduction Velocity (CV)
a. Ideally, around 23 mm/ms for all anterior and posterior electric
b. Ideally, around 8 mm/ms for posterior touch
i. Only lateral fibers get activated
c. Does CV differ?
i. Ideally, your touch/electric anterior and electric posterior should
have been the same
1. This is because you are stimulating MGF with electrical
a. MGF is big, so it’s FAST
2. Additionally, you’re activating MGF with anterior touch
ii. Touch posterior should have been SMALLER
1. You are activating the LGF with posterior touch
a. Smaller, and therefore slower
4. Do thresholds differ?
a. Theoretically, anterior/posterior thresholds should NOT differ
i. Be around 2 V
ii. Since they both activated the MGF
b. Factors that modify threshold:
i. Axon diameter
1. LGF would have a threshold of around 4 - 5 V
2. If we were detecting it..
ii. Placement of electrodes
1. This affects the spread of current
a. The further away the electrodes are, the more
current is needed to stimulate
2. Current is what causes depolarization
5. Rheobase
a. The plateau
i. Doesn’t go to zero since there are always channels that leak out
b. Significance?
i. Usually around the 1 ms mark on the strength-duration curve:
1. The curve flattens out at the Rheobase

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 a. The point where a progressive increase in pulse
duration is NO longer associated with a
progressive decrease in voltage
ii. In other words:
1. For LONGER stimulus durations, the minimal voltage
required to bring the nerve to threshold will be at the
Rheobase
c. Isn’t rheobase a current, not a voltage?
i. Yes, it’s a current
1. BUT, we can convert it...
ii. Given the relationship between current and voltage
1. Ohm’s Law: V = IR
iii. The value of R depends on how WET your worm is
1. Among other factors..
6. Chronaxie
a. Stimulus duration corresponding to TWICE the rheobase value
b. Significance?
i. Given that two nerves have the same Rheobase
ii. The Chronaxie gives an indication of their relative excitabilities
c. How would the strength-duration curve for a set of slow fibers (not
very excitable) compare to the curve for a set of quick fibers (very
excitable?
i. Slower
1. Would be shifted to the right
2. Indicates that for a given stimulus strength, a LONGER
stimulus duration would be needed to bring the slower
fibers to threshold
7. Should be something similar to this plot →
a. Theoretically, it should not have
differed between anterior and
posterior
b. Since the same fiber is stimulated
each time
i. Only stimulating the medial
nerve
1. Much larger
2. Lower threshold!!!
8. Diameter
a. The actual diameter of the giant
fibers is:
i. LGF = 0.05 mm
ii. MGF = 0.07 mm
9. Why did the polarity change when you switched to posterior stimulation?
a. Polarity → shape of the AP waveform (biphasic)
b. It changes because you did NOT move the recording electrodes
between anterior and posterior
i. Recording electrodes were always (-) → (+)
ii. Anode (+) - cathode (-)

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 c. Anterior
i. Up then down
ii. (+) deflection
d. Posterior
i. Down then up
ii. (-) deflection
e. In differential recording, the order of the electrodes changes the shape
of the waveform

10. If you increased voltage, the amplitude of the AP increase?

a. For any neuron, APs are an all-or-none event
i. So, the amplitude would remain the same
b. Theoretically, there COULD be a difference between recruiting the MGF
only vs. MGF + LGF into the CAP if you increase the voltage
11. Why can’t you measure latency with touch stimulation?
a. The SA (stimulus artifact) that
shows up on the trace during
touch
i. Is NOT an artifact from
the touch itself...
ii. It’s from the GRASS
STIMULATOR
b. You therefore don’t know
exactly when you touched the
worm
i. At least not down to the
millisecond
c. So, you can’t measure latency...
12. Propose a modification