Laboratory Exercises Anima

Zoology 120 Animal Physiology
LABORATORY EXERCISES
Midyear Term AY 2015-2016
GENERAL LABORATORY INSTRUCTIONS

1. Read the protocol in advance. Understand
requisition of needed reagents and for
clearly the instructions especially for the
checking the room after the experiment.
use of laboratory apparatus or equipment.
REQUEST FOR THE REAGENTS A WEEK
2. Each
group
will
be
responsible
for
bringing the fresh/live specimens and the
BEFORE THE LABORATORY EXERCISE.

4. Observe cleanliness and orderliness in the
following materials:
laboratory.
Dissecting set
5. Work carefully and follow precautionary
Cleaning rags (cleaning glass and for

wiping mess)
measures.
6. Avoid
Cotton
conducting
unauthorized
experiments.
Alcohol
7. Glasswares will be borrowed from the
Household bleach (chlorox)
Instruments Room.
Detergent /soap
8. Laboratory reports will be passed two (2)
Masking tape small
meetings after the laboratory exercise is
Marker
done. Deadline is 4:30 PM.
Plastic/small garbage bags

Matches
Sponge
Ruler
Pencil
3. A group will be assigned as monitor for
each experiment. The assigned group
monitor
will
be
responsible
for
the
MAINTENANCE OF LABORATORY FACILITIES

1.
Always wash and clean each item used in the experiment.
2.
Return all laboratory materials/ chemicals to their proper places before leaving the room.
3.
Living animals used for experimental work must be treated humanely with utmost
consideration given to minimizing any discomfort that an experiment might entail.
4.
Dispose animals or parts of it properly.
5.
All reusable equipment such as pipettes must be washed and disinfected with a 1:10
solution of household bleach.
6.
Working area of each group must be clean of debris, spilled items and other non-essential
or disposable materials
7.
A laboratory disinfectant should be used to clean laboratory surfaces.
8.
Before leaving, check all electrical and lighting fixtures in order to avoid accidents. Also
make sure that the groups assigned locker is properly closed and secure.
SCHEDULE OF EXPERIMENTS
LABORATORY REPORT FORMAT
Experiment
Abstract
1.
Permeability of the Cell Membrane
I.
2.
Reflex Activity of the Frog
Objectives
3.
Sensory Pathways in Man
Brief background of the experiment
4.
Human Chorionic Gonadotropin
5.
Blood Studies, Pulse and
Summary of procedures
Blood Pressure Determination
In narrative form
6.
Human Respiration Avoiders
7.
Measuring Carbon dioxide Production
Introduction
II. Materials and Methods
III. Results and Discussion
and Oxygen Consumption of Selected
Presentation of data thru figures

(graphs, pictures) and tables
Test Animals
Explanation of results
8.
Osmoregulation
Include
9.
Urinalysis
10.
Characterization of
IV. Answers to Questions
Digestive Enzymes of Killed Animals
V. References
proper
references
citation
of
PERMEABILITY OF THE CELL MEMBRANE
OBJECTIVE
cell membrane.
How the cell controls the
In this experiment, you will learn about:
flow
is
1.
experiments.
Osmosis, a very fundamental process
of
water
shown
in
hemolysis
The osmotic content of the
to cell life
human RBC is about 0.9% sodium chloride. In
2.
dilute or hypotonic solutions, the cells swell
Selective permeability property of the
cell
3.
due to influx of water.

Factors that determine permeability
hemolyze.
During
Eventually the cells

hemolysis,
the
cell
contents including the red colored protein,

INTRODUCTION
hemoglobin,
are
released
into
the
The cell membrane bears the primary
surrounding medium. Hemolysis is indicated
responsibility of determining what materials
by the change in appearance from opaque
come in and out of the cell and at what rate.
red cell suspension to a clear pink solution.
This regulation is very crucial especially for
Increasing the osmotic concentration of the
the
red blood cells by suspending them in a
proper
nutrition,
maintenance
of
irritability of the cells and homeostasis. The
medium
cell
particles results to observable changes. Thus,
membrane
permeable.
is
Its
highly
basic
selectively
structure
containing
permeating
solute
and
the relative rates of permeation of substances
composition as illustrated by the fluid mosaic
can also be determined using rates of
model explains the behavior.
hemolysis as basis.
The cell
membrane transports molecules through it

by
both
passive
and
active
processes.
Permeation of substances may occur through
MATERIALS AND METHODS

Reagents:
the lipid layer or through protein channels or
0.9% NaCl
pumps. The passive mechanisms of transport
0.2M NaCl
depend on available energy from combined
0.1 M NaCl
chemical
0.02 M NaCl
potentials.
and
electrical
0.3M Glucose
and
0.2M Glucose
require metabolic energy expenditure by the
0.1M Glucose
cells. The species-specific proteins and lipids
0.2 M KCl
in the cell membrane spell the differences in
0.1 M KCl
behavior of cells from different organisms.
0.05M KCl
The RBC especially of humans is a convenient
Distilled water
specimen for studying the permeability of the
Neutral red
against
active
means
their
gradients
or
move
substances
The
gradients
Other Materials:
3.
Test tubes
Mix by gently inverting the test tube

from side to side several times.
Test tube rack
4.
Sterile blood lancet
Place the test tubes against a proper

background.
Cotton
5.
Observe hemolysis by looking at the
Paper with prints
prints through the liquid at the sides
Alcohol
of the tube.
Hemolysis is indicated
when prints finally turn clear or when

Technique for drawing blood
the suspension becomes translucent to
1.
the naked eye.
Clean one fingertip of donor with

cotton wet with alcohol; and allow to
2.
3.
of glucose, NaCl and KCl. For each
Unwrap cover of sterile blood lancet
tube, use 5 ml of the test solution with
making sure that the pointed tip is
5 drops of blood suspension. Do
untouched.
hemolysis reading at a fixed time
Hold firmly 3/4 of the body of the
interval.
6.
7.
Use
the
values
you
obtained
to
middle fingers.
calculate the isotonic coefficients of
With your other hand, hold fingertip of
the salts.
donor.
5.
Establish the hemolyzing concentration
dry.
lancet with your thumb, index and

4.
6.
The isotonic coefficient is
the apparent number of ions released

With a swift and forceful stroke, prick
upon ionization of the salt in solution.
the fingertip with the blood lancet.
It
To make a red cell suspension, allow
hemolytic point (or osmotic activity)
10 drops of blood to drip freely into
relative to the non-electrolyte.
can
be
determined
from
its
the test tube filled with isotonic saline

solution
or
until
the
saline
suspensions color becomes reddish

pink.
HEMOLYSIS
1.
Get 2 test tubes. Fill the first test tube

with 5 ml distilled water. To the
second tube, add 5 ml of 0.2 M NaCl.
2.
Add 5 drops of RBC suspension to

each of the 2 test tubes.
Isotonic coefficient =
Hemolytic point of electrolyte (molar concentration)

Hemolytic point of non electrolyte
PENETRATION OF ALKALI
Question for Research
1.
1.
Add 10 ml of neutral red solution to

15 ml of yeast suspension. Take note
chemical factors that affect the process
of any changes in color that may occur
of diffusion.
over a period of 5 min. Neutral red is
2.
2.
What are the factors responsible for
red at pH below 7 but yellow at pH
the
between 7 and 8.
substances (used in the experiment
Measure 2 ml of the mixture and place
required to produce hemolysis?
in 4 labeled tubes. Add the ff:
3.
difference
Discuss
the
Test tube A0.5 ml OF 0.01 N NH4OH
permeablilty
Test tube B0.5 ml of 0.01 N KOH
membranes.
Test Tube C0.5 ml of 0.01 N NaOH

Test Tube D 0.5 ml of distilled water
3.
Cite and explain the physical and
Filter each mixture and observe color

of yeast cells and the filtrate under the
microscope.
4.
in
concentration
factors
of
affecting
the
of
the
plasma
Discuss the penetration of Alkali in

relation to their degree of ionization.
REFLEX ACTIVITY IN TOAD

INTRODUCTION
The reflex arc is the basic unit of an integrated neural activity and consist s of the following: (a)
sensory receptors (b) afferent neurons to transmit impulses to the central nervous system (c) one
or several synapses (d) efferent neurons that sends messages from the CNS and (e) an effector
organ.
In toads, reflexes in response to different stimuli may be described into three general
types (1) simple coordinated movement involving contraction of a relatively small number of
muscles (2) complex coordinated movements involving contraction of a large number of muscles
acting in due sequence to produce definite purposeful motions of a complicated nature and (3)
uncoordinated or convulsive reflexes involving contraction of several muscles acting often in
opposition to another.
The exercise aims to provide the opportunity to observe the different
reflex activities of the toad.

METHODOLOGY
SPINAL REFLEXES: The following observations should be made on normal, spinal and
double pithed toads. Record the observations.
A.
Posture: Place the experimental animal on the desk. Take note of the position of the head,
eyes, forelimbs, hind limbs and the belly. Draw the posture for each of the experimental
animals. Determine the respiration rate by counting pulsation caused by the raising and
lowering of the throat area per unit time. Also, take note of the closing and opening of the
nostrils if any.
B.
Righting Reflex: Place the animal on its dorsal side. Observe the movements of the body
and the limbs and the manner in which it turns to its normal position. Observe swimming
reflex by placing the animal gently in an aquarium or large glass vessel filled with water
room temperature.
C.
Withdrawal Reflex: Suspend the animal on an iron stand by a wire hook passed through
the lower jaw and pinch the left toe with forceps. Observe which leg flexes and extends.
D.
Scratch Reflex: Get a piece of filter paper and moisten with 80% acetic acid. With the
animal still suspended by its lower jaw, place filter paper on the surface of the belly. Take
note of the reaction of the legs toward the irritated spot.
REFLEX TIME: Use the spinal animal utilized earlier.

Prepare beakers, each filled with about 100 ml of the different available concentrations of
HCl. Also prepare a 100 ml beaker with 1% NaHCO3 solution and another beaker of tap water.
Place the long toe in 1% HCl. Make sure you do not let any part of the foot come in contact with
the walls of the dish or acid. Likewise, do not allow the toe to remain in the acid for more than
1.5 mins. Note how many seconds it takes before the foot is withdrawn. Immediately bathe the
foot in 1% NaHCO3 solution and the in tap water. Remove excess water from the foot and toes
with coarse filter paper. Let the animal rest for about a minute. Repeat the experiment using the
different concentrations of HCl prepared. Make sure that the same toe is used and that it is
immersed to the same extent in each trial. Record all results.
INHIBITION OF REFLEXES: Use the spinal toad utilized earlier.
Suspend it by its lower jaw. Determine the reflex time with 0.3% HCl by following the
procedure in the previous experiment using the toe in the other foot. If this solution gives a
reflex time of less than 3 seconds, use a more dilute solution. Determine the reflex time. Bathe
the foot in 1% NaHCO3 and then in tap water. Allow the toad to rest for at least 3 minutes. Again
dip the left toe in the acid, at the same time pinch the toes of the right foot. Take note of the
time the toad moves the left toe. What is the reflex time of the left toe?
Questions for Research
1.
What is the physiological significance of reflexes? Discuss also the need for an intact
nervous system for coordinated activity.
2.
What is decerebrate rigidity and spinal shock?
3.
Discuss the mechanism of reflex inhibition.
SENSORY PATHWAY IN MAN

INTRODUCTION
posterior chamber of the nares. Remove
During the process of evolution, the higher
the tubing, rest for a minute. Place tubing
brain level (cerebral cortex) in man developed
again inside the nasal chamber but make
into a more advanced nerve center.
It is
sure this time that it is in the upper
responsible for motor and sensory functions
anterior chamber. Compare the strength
previously performed by less developed brain
of the sense of smell in the lower and
of lower animals. It also serves primarily as a
upper chambers.
storage area for information and as an organ

of
associative
memory
both
essential
B. Gustatory Sensations
components enabling the animal to profit
Taste Zones: Dry the tongue of your
from past experience. Likewise, a parallel
subject using a clean filter paper and
development and specialization of sensory
place sugar crystals on the tip of the
receptors were attained since an advanced
tongue.
central nervous system would be highly
seconds before the sweet taste is sensed.
dependent
man,
Repeat test but this time, use a drop of
effective contact point between the animal
sugar solution. Again record how long it
and
highly
takes for the subject to taste the sugar.
specialized receptors can be characterized by
Prepare different solutions: 0.5, 1.0, 5, 10,
its sensitivity to a certain stimulus and/or
25 and 50%. Take a small amount of each
sensation and its ability to translate such into
solution and place a few drops on the
mouth of the subject.
its
nerve
on
such
structures
environment.
impulse,
which
as
These
are
in
turn
Take note of the number of
Start with the
transmitted to the appropriate nerve centers
weakest solution. What is the lowest
in the brain.
concentration
The exercise aims to identify
the location and functions of some sensory
of
sugar
solution
that
evoked the sweet taste in the mouth?
receptors.
C. Visual sensations
METHODOLOGY
1. Blind spot
On a piece of paper make two figures.
A. Olfactory Sensations
On the right side make a circle with a 5
Connect one end of the rubber tubing to
mm diameter and on the other side a
the stem of a funnel and place funnel
small cross measuring 1 mm. The two
over a container with an odoriferous
figures should be 60 mm apart. Hold the
substance like perfume or cologne. Insert
paper at arms length in front of the eye
the other end of the tubing into the lower
with the figures arranged directly in front
of the eye. Close the left eye and fix the
bring the watch towards his ear.
right eye upon the cross at the left hand
subject must indicate when he first hears
mark. Move the paper towards the face
the clicking of the watch.
gradually. At a certain distance, the circle
distance of the watch from the ear.
will disappear. Measure this distance and

record it.
The
Measure the
2. Equilibrium
Ask the subject to place the right foot in
2. Pupillary reflex
front of the left foot with the big toe of
Instruct the subject to stand in a place
the latter touching the back of the right
where the lighting is relatively dim or an
foot. Subject must stand upright and be
area far from the window. Note the size
as still as possible. Observe the body
of the pupil in each eye. Then for about
movement. Direct the subject to place
5 seconds, shine a light (use a pen light)
feet together with the sides touching.
into one eye, six inches away from the
Observe the body movements with (a)
eye.
eyes closed and (b) eyes open.
Observe. Remove light;
ask
the
subject to close his/her eyes for two

minutes. As soon as subject opens eyes,
E. Cutaneous sensations
note the size of his pupil. Note if there is
1. Referred pain
constriction or dilation of the aperture of
Place elbow in a large shallow pan of ice
the eye.
cold water and note the progression of
3. Near point accommodation
sensation experienced.
Initially, pain will
Hold a pencil approximately 50 mm from
be felt in the elbow. Later, pain will be felt
your eyes.
elsewhere.
Close the right eye and
gradually move it slowly towards the left
2. Identifying receptors
eye. At a certain distance, the tip of the
Draw 1 cm2 on the back of the hand.
pencil will be blurred and will not produce
Divide it into 16 equal parts. Take a
a clear image. Measure the distance. Test
straight pin and gently touch each square.
the other eye and also measure the
Whenever a sensation is felt, mark and
distance.
record the chart with a p.
Repeat the
same experiment this time using a dull

D. Auditory Sensations
pencil point gently touching the square.
1. Sound Localization
Record on the chart t tactile sensations
Conduct the following test in a quiet
when you feel pressure.
room.
end
Instruct the subject to close his
of
paper
clip.
Straighten one
Hold
the
eyes and plug the left ear with cotton.
straightened end in an ice cube for not
Hold a watch a few feet away from him,
less
but in line with his right ear and slowly
experiment
than
five
by
min.
touching
Again
repeat
each
square
gently. Record your reaction to cold on
less than 3 min. Repeat the experiment.
the chart with a c. Rest hand for 5 min.
Record your reaction to hot on the chart
Carefully, place the straight end of the
with an h.
paper clip in a beaker of hot water for not

1. Differentiate between nerve and bone conduction deafness.
2. How can bone conduction remedy abnormalities?
3. Of what importance are vestibular reflexes? Give other factors involved in equilibrium.
4. Discuss the different types of nerve endings in the skin and their differences in terms of
stimuli, sensitivity and location.
HUMAN CHORIONIC GONATROPIN

INTRODUCTION
Human chorionic gonadotropin (HCG) is a glycoprotein hormone produced in pregnancy that is
made by the developing embryo soon after conception and later by the syncytiotrophoblast (part
of the placenta). Its role is to prevent the disintegration of the corpus luteum of the ovary and
thereby maintain progesterone production that is critical for a pregnancy in humans. HCG may
have additional functions; for instance, it is thought that HCG affects the immune tolerance of the
pregnancy. Early pregnancy testing, in general, is based on the detection or measurement of
HCG.
Human chorionic gonadotropin is a glycoprotein composed of 244 amino acids with a molecular
mass of 36.7 kDa. It interacts with the LHCG receptor and promotes the maintenance of the
corpus luteum during the beginning of pregnancy, causing it to secrete the hormone
progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries
so that it can sustain the growing fetus. Due to its highly-negative charge, HCG may repel the
immune cells of the mother, protecting the fetus during the first trimester.
PROCEDURE
1.
Add the same volume of diethyl ether to your urine sample. The urine should be from a
pregnant female preferably in her first trimester.
2.
Using a burette, extract the urine.
3.
Place in a container, cover and store in the refrigerator while not in use.
4.
To 1 sexually immature female mouse, inject ml of the urine subcutaneously twice a day
for three days. The other mouse will serve as control. Use a 1 ml syringe
5.
Rest on the fourth day.
6.
On the fifth day, sacrifice both mice. Dissect and observe the uterus and the ovary.

1.
What is the function of HCG?
2.
How does HCG induce ovulation in mice?
BLOOD STUDIES, PULSE AND BLOOD PRESSURE DETERMINATION

A. ABO System of blood typing
3. Once a fibrin thread is observed to cling
The agglutination reaction is the basis for
to the pin, take note of the time.
classifying
time when blood leaves the vessel up to
blood into
different
blood
types:
The
the time fibrin is formed is the clotting
1. Draw two circles on a microscope slide.
time.
Mark them A & B.

2. Clean your left hand ring finger with a
cotton ball dipped in alcohol. Dry.
1. Arrange test tubes in the ff series:
3. With a sterile lancet, make a puncture at

the tip of the finger. The lancet would be
able to pierce around 1/8 inch so that
only one puncture is needed
4. Place a drop of blood
C. Factors affecting coagulation

a) Empty
test
tube
for
normal
coagulation
b) Small amount of cotton fibers at the
bottom of the tube
on each of the
c) To be placed on an ice bath
circles on the slide, then add a drop of
d) To be placed in a hot water bath
anti-A serum to the blood in the circle A
e) 1/2 pinch EDTA
and antiB serum to that in the B circle.
f) To be continuously stirred
Stir the blood with separate toothpicks.
2. Collect blood. Without delay, add 2 ml of
What did you observe? For confirmation,
blood to each tube. Remove the needle
observe under the microscope. Obtain the
when transferring blood.
blood types of all members of the group.
3. Observe what happens in each tube. Take
Get the results of the class. How do the
note of the clotting time in each tube.
percentages
compare
with
national
percentages? With those of other races?
D. Pulse Rate
The pulse may be counted in the jugular
B. Clotting time
1. Follow
numbers
but conveniently, the radial artery of the

2
&
of
the
wrist is used. Establish a basal pulse rate
agglutination procedure. Put a drop of
with the subject lying in a flat position.
blood at the center of a clean slide. Take
Make three trials with an interval of one
note of the time when the blood was
minute in between.
shed from the finger.
normal pulse. Determine the
Regard this as the

average
2. Get a needle or pin and draw the blood
pulse rate of three of the following
from the center to the periphery to
subjects. Record and compute for the
observe if fibrin will cling to the pin. Do
average.
this every minute.
AGE:
A. Children 5 to 10 yrs
B. Teenagers 13 18 yrs
1. Why is the red cell number more in males
C. Adults 30 and above
than in females? More in infants than in
SEX: must be 17-22 year-old and non-
adults? Give factors that cause deviation
athletes
from the normal value. Do you consider
A. Male
these normal mechanisms? When do you
B. Female
consider a deviation pathological?
ACTIVITY: 17-22 year-old
2. Is there a difference between white blood
A. Athletes w/ regular training
cell number in males and females? Does
B. Untrained subjects
increase
C. Subjects after 10 min of exercise
proportional increase or decrease in the
OTHERS:
different types of WBC?
or
decrease
denote
A. Pregnant on first trimester
3. Can type O blood be transfused to type
B. Pregnant on last trimester
B? How about the antibodies of type O as

donor, will these not clump the RBC of
E. Blood Pressure
the recipient?
Wrap and secure the rubber cuff of the
4. If a man is Rh positive and the wife is RH
sphygmomanometer on the left arm of
negative, is there a chance for a normal
the subject. Place a stethoscope on the
child? When and how will it happen
artery below the cuff. Listen for the pulse.
physiologically? And medically?
Press the bulb until there is no sound
5. Is coagulation the same as agglutination?
heard on the stethoscope. Deflate the cuff
How does temperature affect clotting
gradually. Take note of the reading in the
time?
meter when the first sound is heard in the
patients to take something cold after a
stethoscope.
tooth extraction
pressure.
This is the systolic blood

As cuff pressure is further
reduced, the sound suddenly becomes

faint. Check the reading and record this as
the diastolic blood pressure. Record the
systolic/diastolic pressure. Repeat blood
pressure determination but this time asks
Why
do
dentists
their
6. What buffers are found in blood plasma?

7. Discuss
the
effect
of
the
different
conditions tested on pulse rate.

8. Discuss other factors that can influence
pulse rate.
9. Explain
the
principle
the subject to do exercises for 30 min.
pressure determination.
Get the blood pressure as soon as subject
10. What is pulse pressure?
stops and also 30 min after complete rest.
advise
behind
blood
HUMAN RESPIRATION
INTRODUCTION
The respiratory center in the medulla oblongata is where information for the need of ventilation
is received and coordinated. It is sensitive to the concentration of carbon dioxide in the blood.
A slight increase in carbon dioxide in the blood produces deeper and faster breathing, permitting
more carbon dioxide to the lungs and consequently the removal of the gas.
This stabilizing
process continues until the carbon dioxide level returns to normal.

Respiration may be divided into three phases: (1) pulmonary ventilation wherein gases (CO 2) in
the lungs are exchanged for gases (O2) in the atmosphere, (2) internal respiration or exchange of
gases between pulmonary capillaries and alveolus and (3) external respiration wherein tissue cells
give up CO2 in exchange for O2 from the capillaries. All three phases depend on the partial
pressure of gases such that simple diffusion occurs.
The aim of the exercise is to observe
different breathing patterns in man.

METHODOLOGY
For parts A-C, each group should choose subjects of the same sex and with similar body sizes.
Do the procedure in part D on a male and female subject.
A.
Normal Respiration Rate

The normal respiration rate, also known as quiet respiration, is measured by counting the
number of breaths per minute while the subject is in a sitting (resting) position. Note the
depth of breathing as shallow, moderate or deep. After recording the normal respiration
rate, instruct the subject to hold tightly a paper bag over nose and mouth. Let him / her
breathe into the bag while recording the respiration rate and taking note of any changes
in the depth of breathing that may occur. Record all data.
B.
Breath Holding Time

Immediately after inhaling, have the subject hold his /her breath for as long as she/he can.
Determine time, in seconds, the subject can hold his/her breath. This is the breath holding
time. Note the depth of breathing and number of breaths while at the same time
observing any changes in the depth of breathing. Record all data.
C.
Hyperventilation
Instruct the subject to breathe deeply with the mouth open (in order to over ventilate the
lungs) for as long as he / she can. The subject may experience difficulty in breathing and
may force himself to continue. Ultimately, cessation of breathing will take place for 10-20
seconds. Record the length of time the subject can force oneself to hyperventilate. Take
note also of the duration or period the subject temporarily ceases to breathe. Likewise,
record the respiration rate and depth of breathing immediately after the recovery period.
Record all data.
CAUTION: HEAVY hyperventilation may cause dizziness or fainting. Limit over ventilation
from 30 to 60 seconds only.
D.
Recording of Respiratory Patterns

Record the respiratory movements of subjects following activities: swallowing water,
reading aloud, laughing, mentally multiplying 789 by 234, coughing and yawning. For the
effects of exercise, have the subject perform 15 to 20 deep knee bends and then
immediately return to his sitting position to record his respiratory movement. Take note of
the time elapsed before respiration was increased and the length of time retained at an
elevated state.

1. Discuss the respiratory center, its control of breathing and factors that may influence it.
2. What is the average normal human respiration rate? What are the factors affecting respiration
rate in humans.
3. What is the effect of forced breathing on the length of time one can hold his breath?
MEASURING CARBON DIOXIDE PRODUCTION AND

OXYGEN CONSUMPTION OF SELECTED TEST ANIMALS
INTRODUCTION
Respiration in aquatic animals is different from that of land animals basically because of the
amount of oxygen available in the water. Concentration of dissolved oxygen is dependent on
factors such as properties of water and the solubility of gases. Essentially, oxygen in the aquatic
environment is less than that found in the air. It is the objective of this exercise to determine the
factors affecting respiration rate in aquatic animals.
METHODOLOGY
Label six 50 ml beakers E1 to E6 and one similar bottle C (CONTROL). Fill up all the bottles with
100 ml of dechlorinated water and add two drops of phenolphthalein indicator.
A.
Respiration rate of fingerlings
Place in each E bottle 2 fingerlings. The C bottle will not contain any animal. Note the start of the
experiment and record the time.
Observe all activities of experimental animals like mobility inside the vessel, gill movements, etc.
from beginning to end.
After half an hour, remove all animals from the bottle and measure
collective weight of the animals. Enter all values in a table. While other members of the group
are weighing the subjects, the rest of the group can proceed and determine the carbon dioxide
content of the water in each of the bottles including the control. Using a base burette, slowly
add 0.4% NaOH to the water while gently shaking or swirling the bottle to ensure thorough
mixing.
Continue this procedure until enough NaOH solution turns the water to a light pink
color. Record the amount of NaOH used. Enter all data in a table. Following the same procedure,
repeat the experiment using the same animals in E1 to E3 but at a temperature 10C below room
temperature.
Likewise do the same for E4 to E6 at a temperature above 10C. Separate control bottles should
be used for separate conditions. Enter all data in the table. Take note of the activity of the
animals in the bottles subjected to different temperatures.
Table 1. Carbon dioxide (moles) produced is equivalent to ml NaOH used.
B.
Measuring Oxygen Uptake
Preparation of the Scholander Respirometer

The Scholander respirometer is a simple respiration or metabolic chamber adapted for use with
small terrestrial animals. Prepare the set up as illustrated below.
Record the weight of the mouse. Place it inside the airtight glass jar. Make sure the mouse does
not touch the NaOH placed underneath the wire mesh as it will cause burns. Introduce a drop of
water on the outer end of the graduated pipette. As the oxygen is consumed by the mouse, CO2
is given off, but this is absorbed by the NaOH. This results in the decrease in pressure inside the
jar causing the drop of water to move inward. Determine the oxygen uptake for one hour at ten
minute intervals by measuring the movement of water in the pipette. Express readings as ml
oxygen uptake per gram body weight per hour.
1. What are the factors affecting respiration rate of aquatic animals? Terrestrial animals?
2. What is the effect of temperature on respiration rate?
OSMOREGULATION
PREPARATION
Collect at least 20 earthworms for this experiment. The best time to hunt for earthworms is on a
damp night when it is not too cold. Use a flashlight to help locate them in patches of grass, in
parks, lawns or fields. You may have plenty in your garden. Usually, you will find them stretched
out on the surface of the ground with their posterior end inside their burrows. They may pop
back into the ground when you appear.
If you collect during the day, you may dig for them preferably at places where the soil is
loose. Digging for worm however, is not equally successful in the different parts of the garden or
field. It is preferable to dig for worms after a heavy rain. Heavy rain drives worms to the surface
where they may be seen crawling on the ground. Actually, worms come up from their flooded
burrows below the ground.
Bring the worms to the laboratory in an uncovered can or glass jar containing some damp soil
and some moist leaves or grass. When you reach the laboratory, tumble them on paper towels to
remove excess soil. In groups of four, place them in separate containers with rain or aged tap
water.
PROCEDURE
1.
Prepare 5 (NaCl) solutions of the following concentrations: 0.03 M, 0.06 M, 0.09 M, 0.12 M
and 0.15 M.
2.
Weigh the worms in groups of four and place them in separate containers with aged tap
water.
3.
Weigh at the end of 15 and 30 minute. If the weights are fairly constant already, determine
the volume of each group by volume displacement in a graduated cylinder containing
water.
4.
After you have determined the weight and volume of each group, transfer each group to a
different dish containing one of the salt solutions.
5.
Weigh the worms at 20 mine intervals within 40 to 100 minutes of immersion. At the end
of the experiment, determine their volume.
6.
Record your results in tabular form, then, (a) plot on graph paper the weight and volume
changes against time and (b) plot the percentage gain or loss in weight and volume
against time and osmotic value of the salt solutions.
QUESTIONS:
1.
What determines the salt content of the soil? What is the effect of rainfall on the soil? On
the behavior of worms? What mechanisms are of survival value to the earthworms in
variable environments?
2.
What response in the weight and volume of the earthworms do the different salt solutions
evoke?
3.
Compare the water exchanges in the worms exposed to varying saline media. What is the
regulatory role of these exchanges?
4.
Why ionic regulation, volume regulation, and osmoregulation are inextricably associated
with each other.
5.
Aside from osmoregulation, what is the other reason for the earthworm to crawl to the
surface at night? Do they have eyes?
URINALYSIS
INTRODUCTION
The basic function of the excretory system is to regulate and maintain the chemical attributes of
the blood. It is also considered a mechanism for maintaining the homeostasis of the internal
medium.
Different organs eliminate different substances and the by-products of metabolism.
The lungs remove carbon dioxide while water and salts are removed through the skin and
kidneys. Salts of heavy metals and blood pigment products are excreted mainly via the kidneys
in urine form.
The examination of urines composition by various tests can more or less
determine the physical state of an animal. It also serves as an index for determining the presence
of metabolites in small or large amounts in the urine. Normal urine is a highly complex solution
of organic and inorganic compounds representing large waste products derived from the
metabolic processes. Urine constituents are chiefly in the form of nitrogen containing and
nitrogen free substances as well as inorganic salts. The principal dissolved substances are urea,
uric acid, ammonia, chlorides, phosphates and sulfates. The exercise will examine the different
physical and chemical characteristics of urine samples.
METHODOLOGY
Collect urine samples.
A.
Physical characteristics
Take note of the following characteristics: color, odor, pH, transparency, odor, amount.
Then vigorously shake the samples and take note of any changes. Enter all data in the data
sheet.
B.
Chemical Characteristics of the urine
1.
Albumin Test:
Filter the urine through coarse filter paper to clean any turbid specimens that may be
present. Heat 5 ml of urine in a test tube to boiling. Note the color of the precipitate
formed, which is caused by albumin and phosphates. If no precipitate is formed, the
sample is negative for albumin. Acidify the resulting hot mixture by slowly adding 3-5
drops of acetic acid that will result in the clearing of the precipitation caused the presence
of phosphates. If the samples remain clear, albumin is absent, otherwise the precipitate
will persist and the mixture may become more flocculent.
2.
Glucose Test:
If albumin is present in appreciable amount, it should be removed by acidifying the urine
with dilute acetic acid, boiling and then filtering. The test is then performed on the filtrate.
Place 5 ml of Benedicts solution in a test tube and add to it 0.5 ml of urine sample. Boil
vigorously for 1-2 min over an open flame and allow it to cool slowly. If the urine remains
clear or white turbidity develops (presence of ureates or phosphates) no sugar is present.
But in the presence of glucose, the entire solution becomes opaque and filled with
precipitate colored green to red indicating the amount or quantity of sugar present.
Positive result may be recorded based on the following:
0.1 to 0.25% glucose indicated by green turbidity (weak)
0.5 to 1 % glucose indicated by yellow to orange precipitate (moderate)
> 1 % glucose indicated by brick red precipitate (strong)
3.
Urea Test:
Evaporate about 3 ml of urine to complete dryness, finishing the process in a hot water
bath. Remove the sample from the water bath by rubbing the residue with enough
acetone (using a stirring rod on a watch glass) Allow cooling. Crystals appearing as crystal
needle or the like indicate the presence of urea.
If necessary, examine a drop of the
treated samples under the microscope in order to determine the presence of urea crystals.
4.
Uric Acid:
Treat 10 ml of urine with 2 drops of strong NH4OH. Then saturate the solution with
powdered NH4Cl in order to settle the mixture. Pour off the resulting supernatant liquid
into another beaker. Take note of the precipitate that may indicate ammonium urate and
transfer it to a clean evaporating dish. Add 2-3 drops of strong nitric acid and evaporate
to dryness in a water bath. Take note of the color of the resulting residue. Add 2 ml of 1%
NH4OH. Again take not of the resulting color. The presence of the uric acid positively
indicated by the resulting pink residue upon the addition of strong nitric acid, then turns
purple upon addition of 1% NH4OH.
5.
Indican Test:
Fill a test tube with 10 ml sample and add 5 ml Obemayers reagent. Warm the mixture slightly
and put 3 ml of chloroform. Mix the resulting solution by inverting the test tube from time to
time. Normal amount of indican will give a faint blue color while excess amount of indican will
show an indigo-blue color (chloroform) which sinks to the bottom of the tube.

1. Give the normal physical characteristics (color, transparency, volume, odor, pH) of urine. What
are the substances responsible for these?
2. What pathological conditions are prevailing if the test samples show significant differences in
amount? Composition?
3. What is hemodialysis? Peritoneal dialysis?
CHARACTERIZATION OF DIGESTIVE ENZYMES OF KILLED ANIMALS

Enzymes are needed for the chemical digestion of food. There are specific enzymes for different
types of food. It is thus easy to demonstrate enzyme activity using different substrates.
Methodology:
I.
Preparation of Substrates
A.
Amylase Substrate
Mix 200 mg soluble starch with a little water and make up a volume of 100 ml. Heat the
mixture until it boils and then slowly adds 2 g agar powder. After cooling to about 50
60C, pour into Petri dishes and add 2 drops strong iodine solution, stirring to ensure even
distribution of the iodine and the formation of a uniformly blue-colored gel.
B.
Protease Substrate
Mix 100 ml of milk powder with 2 grams agar powder and slowly heat the mixture until
it boils. Pour into petri dish. Solidify.
C.
Lipase Substrate
Mix 4 g mayonnaise with 5 ml water and add 1 ml of 1 molar NaOH. Prepare the agar
(2%) and allow to cool to 50-60 C. Add 1 ml mayonnaise mixture, stirring to ensure even
distribution. Pour into petri dish.
II.
Determining the Presence of Enzyme Activity

After allowing the gels to cool and harden, cut 3 uniformly sized wells in each plate. Into
each well place uniform lengths of the stomach, small intestines and large intestines of a
freshly killed mouse. Add a little water into each well. If specific enzymes are present, their
diffusion from the wells into the surrounding agar causes digestion of substrate resulting in
the formation of transparent or pale colored zones around the perimeter of the wells. The
total area of a zone developed around each well in unit time is directly proportional to the
concentration of the enzyme, provided factors like temperature and rigidity of the gel are
controlled.
A.
Amylase:
Digestion of starch from the plates leads to the formation of transparent zones around the
perimeter of wells. Plate containing starch-iodine complex need not be incubated.
Satisfactory results can be obtained after 6 hrs at room temperature.
B.
Protease:
Protease digests casein (milk protein) from the gel, forming transparent zones. Plates
should be incubated at room temperature for about 12 hours before they are examined.
C.
Lipase:
Plate should be incubated at 37 C before being examined. Lipase will digest the substrate
to form transparent zones, but this can be seen more distinctly by flooding the plates with
10% copper sulfate solution which should be left for 30 min to penetrate the gel. Zones
containing fatty acids stain blue green after this treatment.
Preparing the animals: Always treat your lab animals humanely. All animals used for this study
must be freshly killed. After killing the animal, transfer it to a dissecting pan and dissect out the
digestive system as fast as soon as you can. The digestive mucosa degenerates very fast so it is
important that you work rapidly. Dissections should be made dry, since enzymes diffuse through
water to spread to regions of the gut where they are not normally present.
Questions for research:
1. What are the different factors that affect enzyme activity?
2. What substances were digested in which part of the digestive tract?

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Zoology 120 Animal Physiology

GENERAL LABORATORY INSTRUCTIONS

requisition of needed reagents and for

clearly the instructions especially for the

checking the room after the experiment.

use of laboratory apparatus or equipment.

REQUEST FOR THE REAGENTS A WEEK

bringing the fresh/live specimens and the

BEFORE THE LABORATORY EXERCISE.

5. Work carefully and follow precautionary

Cleaning rags (cleaning glass and for

7. Glasswares will be borrowed from the

Household bleach (chlorox)

8. Laboratory reports will be passed two (2)

Masking tape small

meetings after the laboratory exercise is

done. Deadline is 4:30 PM.

Plastic/small garbage bags

MAINTENANCE OF LABORATORY FACILITIES

Always wash and clean each item used in the experiment.

Dispose animals or parts of it properly.

solution of household bleach.

Zoology 120 Animal Physiology

A laboratory disinfectant should be used to clean laboratory surfaces.

LABORATORY REPORT FORMAT

Permeability of the Cell Membrane

Reflex Activity of the Frog

Sensory Pathways in Man

Brief background of the experiment

Human Chorionic Gonadotropin

Blood Studies, Pulse and

Blood Pressure Determination

Human Respiration Avoiders

Measuring Carbon dioxide Production

II. Materials and Methods

III. Results and Discussion

and Oxygen Consumption of Selected

Presentation of data thru figures

IV. Answers to Questions

Digestive Enzymes of Killed Animals

Zoology 120 Animal Physiology

PERMEABILITY OF THE CELL MEMBRANE

How the cell controls the

In this experiment, you will learn about:

Osmosis, a very fundamental process

The osmotic content of the

human RBC is about 0.9% sodium chloride. In

dilute or hypotonic solutions, the cells swell

Selective permeability property of the

due to influx of water.

Eventually the cells

contents including the red colored protein,

The cell membrane bears the primary

surrounding medium. Hemolysis is indicated

responsibility of determining what materials

by the change in appearance from opaque

come in and out of the cell and at what rate.

red cell suspension to a clear pink solution.

This regulation is very crucial especially for

Increasing the osmotic concentration of the

red blood cells by suspending them in a

irritability of the cells and homeostasis. The

particles results to observable changes. Thus,

the relative rates of permeation of substances