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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Questions for Research
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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Questions for Research

1.

What is the function of HCG?

2.

How does HCG induce ovulation in mice?

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

AGE:
A. Children 5 to 10 yrs

Questions for Research

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Questions for Research

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?

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.
Questions for Research
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

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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?

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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?

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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.

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

Questions for Research

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?

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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:

Zoology 120 Animal Physiology

LABORATORY EXERCISES
Midyear Term AY 2015-2016

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?