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Animal Physio LT1 Reviewer Lab
Animal Physio LT1 Reviewer Lab
Exercise 1
Dissolution
●
● Results
○ Which substance(s) dissolved?
○ Which substance(s) did not?
○ How can you tell?
Filtration
●
● Results
○ Which substances passed through the selectively permeable disk?
○ Which substances did not?
○ Why do some substances pass through the filter paper while other substances do not?
Diffusion
● Methodology
● Prepared 5% agar and poured onto 6 petri dishes in equivalent amounds
● After agar was set, a well was created in the center of each petri dish
● Prepared dyes
○ methylene blue
■ 100%
■ 50%
○ Congo red
■ 100%
■ 50%
● Filled wells with dye
○ A
■ Petri dish 1: 100% methylene blue
■ Petri dish 2: 100% Congo red
○ B
■ Petri dish 3: 100% methylene blue
■ Petri dish 4: 100% Congo red
○ C
■ Petri dish 5: 50% methylene blue
■ Petri dish 6: 50% Congo red
● Setup
○ A
■ Petri dish 1: Room temperature
■ Petri dish 2: Room temperature
○ B
■ Petri dish 3: Refrigerated
■ Petri dish 4: Refrigerated
○ C
■ Petri dish 5: Room temperature
■ Petri dish 6: Room temperature
● After finishing set-up, time was recorded
● Petri dishes were checked 10 times within 3 days to observe for diffusion and the following were
done in a table:
○ time and date were recorded
○ measured distance from the edge of well to the distance of the edge of diffusion (in mm)
● Calculations and Analysis
○ Rate of linear diffusion was computed in mm per hour
○ Created line graph comparing the three setups
■ X-axis: time in hours
■ Y-axi: rate of linear diffusion
● Results
○ Factors affecting diffusion
■ Temperature
● increasing temperature increases movement, allowing diffusion to take place
● all forms of motion is influenced by heat energy
● heat has the ability to cause random motion in microscopic particles such as
atoms and molecules
● increase in temperature, increases rate of diffusion
■ Molecular Weight/Particle Size
● Congo red (696.68) diffused faster than methylene blue (319.86)
● methylene blue should have diffused faster because of its smaller molecular
weight and particle size
● results reflect that factors other than molecular weight had more bearing on
diffusion (see membrane permeability)
● increase in molecular weight, decreases rate of diffusion
■ Membrane Permeability
● agar = 5% agar + 95% water
● Congo red is water soluble
● methylene blue is slightly soluble in water
● Since agar is mostly water, and methylene is more hydrophobic, it would diffuse
slower despite having a smaller molecular weight
● increase in membrane permeability, increases rate of diffusion
○ in this case, increase in water solubility, increases rate of diffusion
■ Concentration
● 100% concentrated dyes diffuse faster than less concentrated ones
○ 100% concentrations of methylene blue and congo red have 0% water
○ 5% agar has 95% water
○ dyes are more concentrated than agar (agar has more water)
○ well containing dye is concentrated so dye moved to regions with less
concentration and more water (moved away from the well and toward
the agar)
● 50% concentrated dyes contain 50% water
○ diffused slower into the 5% agar containing 95% water
● increasing concentration, increases rate of diffusion
Osmosis
● Concept of osmosis
○ diffusion of water through a semi-permeable membrane
○ water moves from lower solute concentration to higher solute concentration
○ water moves from higher solvent concentration to solvent water concentration
○ movement occurs until equilibrium is reached
○ solutions
■ hypotonic
● solute concentration is higher outside
● results in cell bursting
■ hypertonic
● solute concentration is higher inside
● results in cell shrinking
■ isotonic
● solute concentration is equal
● equilibrium
● Methodology
○ cellophane represented the semipermeable membrane
● Results
○ increase in solute concentratation, increases fluid displacement
■ increase in solute concentration is equivalent to the increase in concentration gradient
■ higher concentration gradient, slows down equilibrium
Hemolysis
● Concepts
○ hypotonicity
■ higher concentration of solute outside cell, a lot of water inside cell
■ cell tends to burst
■ principle behind hemolysis
○ hemolysis
■ clear solution = complete hemolysis
○ isotonic coefficient
■ molarity at which non-electrolyte (glucose) completely hemolyzed rbc ÷ molarity at which
electrolyte (sodium chloride) completely hemolyzed rbc
■ amount of salts to be added to distilled water to make the solution isotonic for rbc
○ degree of dissociation
■ the fraction of the original solute that has dissociated
● Methodology
● Results
○ hemolysis was observed in setups with:
■ calcium chloride (CaCl2)
■ potassium chloride (KCl)
○ hemolysis decreases as dilution decreases
■ increase in dilution causes increase in hypotonicity, increasing hemolysis
○ 2 ions completely dissociated when the isotonic coefficient is 3
Fermentation
Concept: The formation of gas by fermentation produces pressure in the tubes causing the liquid to be pushed.
Methods:
· Fermentation tubes were given mixtures:
o A - 15 ml of 10% glucose solution
o B - 7.5 ml of 10% glucose solution and 7.5 ml yeast suspension
o C - 15 ml sucrose solution
o D - 7.5 ml of 10% sucrose solution and 7.5 ml yeast suspension
o E - 15 ml yeast suspension
· The rate of fermentation was measured as the amount of gas displaced in the tube (measured by cm using
ruler) and were measured at different time intervals of 10 mins, 15 mins, 30 mins, 1 hr, 2 hrs, and 24 hrs.
o A and C did not ferment as there was no yeast
o Change in the pressure of the tubes results when yeast ferments sugars. Production of carbon dioxide pushes the
solution. Foaming was also produced due to carbon dioxide.
o E should not have fermented since supposedly no sugars should be found.
*Error: Residual sugars were found. Tubes were not cleaned properly
o B (containing glucose) fermented faster than D (containing sucrose) because yeast fermentation converts simple
sugars (glucose) to ethanol and carbon dioxide. A disaccharide, has to be first degraded into its monosaccharide
units before it proceeds to fermentation.
o The fast rate of fermentation of E must have occurred since E contained the most amount of yeast.
· Additional test for acidity. 5 drops of phenol red was added then gently shook. The color change of the solution
was:
o A - Transparent yellow
o B - More opaque yellow separated from a darker brown liquid
o C - Transparent yellow
o D – Most opaque yellow
o E - More opaque yellow separated from a darker brown liquid
Phenol red is a pH indicator that turns red at or above pH 7 (alkaline) and turns yellow at a pH lower than 7
(acidic). All setups produced a yellow color indicating their acidic nature. Carbon dioxide from fermentation reacted to
components of the solution which yield to acids such as carbonic acid.
Exercise 2
Brainstem Reflexes
● Methodology
○ Pupillary Reflex
■ shine light on one eye and observe pupil of that eye
○ Consensual Pupillary Reflex
■ shine light on one eye and observe pupil of that other eye
○ Ciliospinal Reflex
■ pinch neck and observe reaction of eyes
○ Corneal Reflex
■ suddenly touch cornea of eye and see what happens
○ Orbicularis Oculi Reflex
■ surprise person by flashing light on their eyes and observe reaction
○ Auditocephalogyric Reflex
■ surprise person with loud sound and observe reaction
○ Gag Reflex
■ prod pharyngeal region of neck and see what happens
● Results
○ Pupillary Reflex
■ result: pupil constriction
● pupil’s role is to allow light to enter retina
● constriction happens when there is too much light to protect photoreceptors
■ anatomic components
● CN II
○ Optic nerve
○ sensory nerve for eye region: responsible for detecting stimuli
● CN III
○ Oculomotor nerve
○ motor nerve: responsible for effector
○ Consensual Pupillary Reflex
■ result: pupil constriction
● though unexposed to light, also constricts so response is consensual
● optic tract conducts visual information from both eyes and the pretectal area
projects bilaterally to both Edinger-Westphal nuclei
○ pretectal area
■ narrow, transversely oriented rostral zone of the
mesencephalic tectum
■ contains several nuclei that receive fibers from the optic tract
■ has bilateral efferent connections with the Edinger-Westphal
nucleus of the oculomotor nuclear complex by way of which it
mediates the pupillary light reflex
○ Edinger-Westphal nuclei
■ contains preganglionic parasympathetic (visceromotor)
neurons whose axons end in the ciliary ganglion
■ Input to the Edinger-Westphal nucleus arises from a cell group
called the pretectum, a cell complex that receives retinal input
and is part of the pathway involved in reducing the size of the
pupil upon light stimulation of the retina
■ anatomic components
● CN II
○ Optic nerve
○ sensory nerve for eye region: responsible for detecting stimuli
● CN III
○ Oculomotor nerve
○ motor nerve: responsible for effector
○ Ciliospinal Reflex
■ result: pupil dilation
● eyes dilate in response to pain: defense against noxious stimuli
● person becomes alert to sensations of pain in neck
■ anatomic components
● CN II
○ Optic nerve
○ sensory nerve for eye region: responsible for detecting stimuli
● CN III
○ Oculomotor nerve
○ motor nerve: responsible for effector
○ Corneal Reflex
■ result: blinked
● blinked in response to tactile stimulation as defense mechanism
■ anatomic components
● CN II
○ Optic nerve
○ sensory nerve for eye region: responsible for detecting stimuli
● CN III
○ Oculomotor nerve
○ motor nerve: responsible for effector
○ Orbicularis Oculi Reflex
■ result: blinked
● defense to noxious stimuli
■ anatomic components
● CN II
○ Optic nerve
○ sensory nerve for eye region: responsible for detecting stimuli
● CN III
○ Oculomotor nerve
○ motor nerve: responsible for effector
○ Auditocephalogyric Reflex
■ result: head and eyes turned toward direction of stimulus
● defense mechanism: concept of ‘arousal’ from psychology
● arousal: type of bodily energy which primes or prepares us for emergency action
■ anatomic components
● CN IV
○ Trochlear nerve
○ motor nerve: responsible for eye movement
○ directed eye to seek stimulus (loud sound)
● CN VIII
○ Vestibulocochlear nerve
○ sensory nerve: for hearing and balance
● CN XI
○ Spinal accessory nerve
○ motor nerve: responsible for head movement
○ Gag Reflex
■ result: gagged
● defense mechanism: discourage organism from eating incompatible substances
(allergenic, toxigenic)
■ anatomic components
● CN IX
○ Glossopharyngeal nerve
○ sensory nerve: responsible for detecting stimuli in pharyngeal area
● CN X
○ Vagus nerve
○ sensorimotor nerve: responsible for sensory and motor functions in
viscera
○ triggers regurgitation
Reflex
● basic unit for behavior is the reflex
● generally occur quickly and are the result of excitation of
○ sensory receptors
○ afferent nerve fibers
○ integration by the central nervous system
○ transmission of motor impulses over efferent (motor) nerve fibers
○ excitation of an effector organ /tissue.
● spinal cord may act as the primary integrator and source of a motor nerve response (spinal reflex) or as a
modulator of motor responses when the brain or higher regions of the spinal cord are involved
● integrating and modulating activity on reflex activity is observed best when the controlling influences of the
brain are removed (why pithing is needed)
Single pithing
● ablation of brain
● insert the scissors so they are back against the joint between the upper and lower jaws before cutting
● effectively breaks all connections between the brain and the spinal cord, it therefore eliminates any influence
the brain could have on reflex activity
Double pithing
● ablation of spinal cord and brain
● destroy the spinal cord by insertion of a mental dissection probe/needle into the open vertebral canal by the
single pith
Muscle Tonus
Electroencephalography (EEG)
epinerium
● loose connective tissue sheath
fascicles
● smaller bundles of axons within epinerium
● each of which is also encased in perineurium
perineurium
● more structured epithelial sheath
endoneurium
● each individual axon is surrounded by a very thin individual connective tissue sheath
toad’s sciatic nerve
● consists of only a single bundle of fibers, surrounded by the perineurium and loose epineurium
Compound nerve
● ex. sciatic nerve
● typically contains efferent (α and γ motor axons, and post-ganglionic autonomic axons) and afferent
(sensory) axons
● individual axons vary in diameter, myelination, excitability, threshold and conduction speed.
Stimulus artifact
● display initially will show only a brief biphasic deflection.
● results from the instantaneous spread of passive electrical current from stimulating to recording electrodes
by the electrolyte on the surface of the nerve
Refractory Period
● during the action potential, a second stimulus will not produce a second action potential.
● we can give the sciatic nerve two stimuli successively, and the intensity of the two stimuli is identical
● If the first stimulus can produce an action potential, then the second stimulus can also produce action
potential, if the interval of the two stimuli exceeds the refractory period of the action potential arising from the
first stimulus
○ At the beginning, the interval of the tow stimuli is very long and exceeds the refractory period of the
first action potential arising from the first stimulus considerably. Under this circumstance, both the
two stimuli can produce action potential
○ Then, we minish the interval gradually, at first, we can record two action potential
○ When the internal is short enough that it equals the refractory period of the first action potential, the
second stimulus can’t produce action potential, and the interval of this two stimuli is the refractory
period of the action potential.
Conduction Velocity
● d/t --where d = the conduction distance; --and t = the conduction time.
● Study Questions
1. How does a CAP differ from a single action potential?
● A compound action potential is the sum of multiple axons in a nerve firing, while a single action potential is
generated by just one axon.
● single action potential (recording from a single axon) is an “all or none” response
○ under normal circumstances, the amplitude of the response does not change with stimulus intensity
○ conduction velocity of individual action potentials increases with axon diameter
■ action potentials of the largest axons will reach the recording electrodes first
○
● compound action potential (recording from many axons) is a graded response
○ magnitude increases with intensity of stimulation
○ this is because different axons have different thresholds of excitation
■ largest axons have the lowest thresholds of excitation and are the most excitable
○ at low stimulus intensities, only the largest axons are activated
○ as stimulus intensity is raised in steps, more and more smaller axons are progressively recruited
●
2. Action potentials are said to be all or none responses. Why does the frog sciatic nerve give a graded
response?
● The frog sciatic nerve is made up of different axons, each with their own threshold. Some are higher than
others while some are lower. Therefore, as you increase the voltage, more and more of them fire, giving a
seemingly graded response
● This graded response phenomenon illustrates the differences in threshold that exists among the different
sizes of fibers that make up the nerve. Remember, you are recording from a nerve, a large bundle of
neurons, each with a different threshold. If the stimulus voltage is increased slowly and smoothly, you may
observe discrete jumps in the amplitude of the compound action potential as different threshold classes of
nerve fibers are “recruited”. As you increase the amplitude more neurons reach their threshold and
contribute to the increase in size of the compound action potential. Eventually, as the stimulus voltage is
increased, a point will be reached when the wave form of the action potential stops changing. At this point all
the fibers in the nerve able to respond to the stimulus are being stimulated. This is a maximal response.
3. What was the smallest voltage required to produce the maximum (largest) CAP? What proportion of the
nerve fibers were excited to produce this maximal response?
● To produce a maximum response, the whole nerve must be stimulated.
● If the stimulus voltage is increased slowly and smoothly, you may observe discrete jumps in the amplitude of
the compound action potential as different threshold classes of nerve fibers are “recruited”. As you increase
the amplitude more neurons reach their threshold and contribute to the increase in size of the compound
action potential. Eventually, as the stimulus voltage is increased, a point will be reached when the wave form
of the action potential stops changing. At this point all the fibers in the nerve able to respond to the stimulus
are being stimulated. This is a maximal response.
4. In this exercise, you examined the effect of increasing stimulus intensity on the nerve. What other stimulus
parameter might also affect the nerve’s tendency to generate a CAP?
● Frequency and duration can affect the nerve’s tendency to generate a CAP; frequency in that if there’s the
right amount of time between them, their effects can be additive and trigger a CAP; duration in that even a
weak stimulus can potentially trigger a CAP if kept up long enough
5. Explain the difference between the relative and absolute refractory periods.
● No action potentials can be fired during the absolute refractory period since too many Na+ channels are
voltage inactivated from the last action potential being fired. However, during the relative refractory period,
enough Na+ channels have opened back up to allow another action potential to be fired, though a stronger
stimulus than normal will be required.
6. Briefly describe the cellular events responsible for the refractory period. (Hint: Discuss the mechanism of
repolarization.)
● After an action potential is fired, a large fraction of the cell’s Na+ channels are voltage inactivated,
preventing a second action potential from being fired. This is the absolute refractory period. The cell then
releases K+, repolarizing the membrane and causing the Na+ channels to open back up. Between the
absolute refractory period and reaching the normal resting membrane potential comes the relative refractory
period, which is explained above.
7. Based on your calculation for CAP conduction velocity, how long would it take the CAP to travel the length of
the sciatic nerve? Assume a total length of 10 cm.
● The conduction velocity of the action potential is determined by measuring the distance traveled (length of
the nerve in m) and dividing by the time (sec) taken to complete the reflex arc, also called the latency.
● Conduction velocity = distance (m)/time (sec).
0.1 m / t = 155.4 m/s
t = 0.1 m / 155.4 m/s = 6.44x10- 4 seconds
Exercise 3
○ Location in Frog
2. How does an action potential move in a bundle of neurons like the sciatic nerve?
● compound action potential (recording from many axons) is a graded response
○ magnitude increases with intensity of stimulation
○ this is because different axons have different thresholds of excitation
■ largest axons have the lowest thresholds of excitation and are the most excitable
○ at low stimulus intensities, only the largest axons are activated
○ as stimulus intensity is raised in steps, more and more smaller axons are progressively recruited
5. What is tetanus?
● infection of the nervous system with the potentially deadly bacteria Clostridium tetani (C. tetani)
● infection begins when the spores enter the body through an injury or wound
● often begins with mild spasms in the jaw muscles (lockjaw)
● tetany: sudden, powerful, and painful contractions of muscle groups caused by prolonged muscular action;
may lead to muscle tears and fractures
Tetanus
muscle contraction
● results in development of tension or force usually measured in grams
● muscles will shorten if they develop more force than the force that is opposing them
● ex: contracting muscles in our arm will shorten and allow us to lift a book if the force developed by the
muscles is greater than the weight (force) of the book.
no tetanus
● stimuli are applied at a frequency where the time interval between stimuli is longer than the time it takes for
the muscle fibers to completely contract and relax, then no tetanus (no temporal summation) is observed
partial/incomplete tetanus
● stimulus frequency was increased until the muscle fibers no longer had time to completely relax
● partial relaxation between muscle twitches
● most sustained voluntary skeletal muscle contractions are incomplete tetanic contractions with different
motor units stimulated at different times (asynchronous contractions)
● asynchronous contractions delay muscle fatigue, which is an inability to contract caused by long periods of
muscle contraction
● stimuli is applied at a frequency where the time interval between stimuli is shorter than the time it takes for
the muscle fibers to completely relax, then partial (incomplete) tetanus is observed
● type of wave summation with partial relaxation observed between twitches
fused/complete tetanus
● stimulus frequency was increased further so that the muscle fiber could not even begin to relax
● sustained contraction with no relaxation observed between twitches
● stimuli are applied at a frequency where the time interval is shorter than the time it takes for the muscle
fibers to even begin to relax, then fused (complete) tetanus is observed
● type of wave summation with no observable relaxation between twitches
twitch contraction
● type of muscle contraction, but not normal muscle contractions
● single, brief stimulus is applied to a muscle fiber, either naturally in the form of a nerve impulse, or artificially
in the form of an electrical stimulus
● quick shortening observed in a skeletal muscle when a single action potential traveling down a motor neuron
stimulates the skeletal muscle fibers of the motor unit to contract
● single contractile event in response to single action potential
latent period
● lasts about 2 msec (milliseconds)
● time between stimulation of muscle cells and force generation
contraction period
● lasts about 10–100 msec
● period during which force (measured in grams) is increasing
relaxation period
● which lasts 10–100 msec
● period when force is decreasing
● phase of contraction; period during which more crossbridges detach than reattach to thin filaments
threshold stimulus
● minimal stimulus that results in a muscle twitch
maximal stimulus
● stimulus that produces maximal force is called the maximal stimulus
● stimulus to the muscle greater than maximal does not produce a greater force
wave summation
● if the muscle fibers of a motor unit are stimulated before the relaxation phase of a muscle twitch is complete,
then the next contraction will produce a greater force
● how to increase force generated
● increasing the frequency of muscle stimulation produces sustained force generation
● increasing the number of motor units contracting at the same time, motor unit recruitment increases force
generated
fatigue
● an inability to contract caused by long periods of muscle contraction
wave/temporal summation
● force of contraction seems to increase like the sum of the individual contraction waves
load
● force that the muscle is contracting against.
displacement
● strength of contraction
● sum of the force exerted by all the motor units that are excited
recruitment
● multiple motor unit summation
● increasing the number of motor units that are stimulated to contract
motor unit
● motor neuron and all the muscle fibers it innervate
Fatigue
Electromyography
Smooth muscle
2. What effect does stretching the muscle have on contraction strength? Is this effect linear? What preload
force resulted in the highest contraction force?
3. What effect does varying the stimulation frequency have on contraction force? Which stimulus interval
caused the greatest contraction force?
5. At what time point did your muscle begin to fatigue? Calculate the % decrease in contraction force by
comparing the force at the end of the experiment with the maximal contraction force.
6. In your own words, explain a possible mechanism for why the muscle was unable to maintain a prolonged
contraction in this experiment. .