FOREWORD

The laboratory manual in Biochemistry is designed to provide students the basic

principles and concepts in Biochemistry. This manual contains a series of experiments,
laboratory techniques, and analytical methods intended to a large group of students of
differing backgrounds. The contents of the manual are simplified to attract or attain
interest of the students. Also, it would like to increase appreciation of the actual
performance of the experiments. The experiments are presented in a very interesting
way. These experiments do not require sophisticated equipment and instruments to be
carried out. These experiments are designed to reinforce whatever concepts and
principles that are being discussed in the lecture. Special topics are included to suit
students’ chosen professional courses.

It is the authors’ hope that students taking up this course would be able to work
in biochemical related problems using appropriate experimental approaches and that
this course in Biochemistry will be of great value to them later in their professional
work.

The Authors
 TABLE OF CONTENTS

EXPERIMENTS PAGES

1 Cell Macromolecules 15

2 Translocation of Materials 19

3 pH and Buffers 25

4 Carbohydrates 30

5 Lipids 36

6 Nucleic Acid 43

7 Proteins 48

8 Enzymes 55

9 Digestion 61

10 Nutrition 71

11 Vitamins 72

12 Pregnancy Test 75

13 Blood 77

14 Urine 84
 SAFETY PROCEDURES AND PRECAUTIONS

General Rules for Chemistry Laboratories

WHEN INSIDE A LABORATORY, DO NOT PUT ANYTHING IN YOUR
MOUTH.
1. Do not eat or drink in the laboratory.
2. Never taste or touch chemicals with the hands unless specifically instructed to do
so.
3. Mouth pipeting is prohibited.

PROTECT YOURSELF AND YOUR BELONGINGS FROM DAMAGE

1. Do not bring coats, books or other unnecessary materials inside the laboratory.
Use lockers.
2. Bare feet and sandals are not allowed. Wear shoes.
3. Do not wear loose clothing. Long hair is potentially dangerous and should be
tied back.
4. Do not wear contact lenses since they are potentially dangerous in a laboratory
with an atmosphere of fumes.
5. In general, all chemical operations should take place in a fume hood.
6. Wear appropriate eye protection. When using laboratory chemicals, equipment
under vacuum or elevated pressure and glassware, wear chemical splash
goggles.

PROPER CARING DURING LABORATORIES SHOULD BE DONE TO REDUCE

HAZARD

1. Label all reagents with appropriate name, date of preparation and initials of
maker.
2. Never put a pipette into a reagent bottle and never return unused solutions or
chemicals to a bottle. Contamination can ruin present and future experiments.
3. Gently place used pipettes, tip down into the marked containers with
disinfectants to control contamination.
4. Rinse all glasswares and remove tape labels before putting them in dirty
glassware dishpans.
5. Hazardous materials are put in specially marked containers according to
instructions.
6. Make sure to clean the area when work is finished and make sure to check that
everything is turned off and put away.
7. Exits and aisles must be kept open.
 Laboratory Rules and Safety Procedures

Safety regulations are strictly enforced in the chemistry laboratory. The rules are listed
below, with the first-aid procedure for common laboratory mishaps.

1. Safety goggles must be worn at all times.

2. Contact lenses are not permitted.
3. Wear proper clothing-no shorts, halter-tops, or sandals.
4. Long hair must be tied back.
5. Smoking, drinking, or eating is not allowed.
6. Sitting in laboratory benches is not allowed.
7. Backpacks and book bags should be left in the hall.
8. Dispose of all waste materials as directed.
9. DO NOT PIPETTE BY MOUTH. Use a rubber bulb or pipette pump for suction.
10. Never point the open end of a test tube toward anyone.
11. Reactions, which produce objectionable odors, must be run in the hood.
12. When inserting glass into a stopper, moisten both glass and stopper first.
Protecting your hands with a towel, grip the glass about an inch above the
stopper and push with slight rotation. (Failure to observe this procedure leads to
more lab accidents than any other single factor.)
13. Reagents bottles:
o Read the labels carefully.
o Do not contaminate reagent bottles.
 Transfer what you need into a beaker.
 Do not insert anything into a reagent bottle.
 Never return unused chemicals to reagent bottles.
o Do not take more reagent than is required for an experiment.
14. Report all injuries to the lab instructor or stockroom personnel.
15. Know the location and operation of all safety equipment, fire extinguishers,
showers, and eye washes.
16. NEVER pour water into concentrated acid. Pour acid slowly into water with
constant stirring.
17. NEVER smell a chemical by putting your nose into a container.
18. After experiments, turn off gas and water, and clean desk and balance.
19. Performing unauthorized experiments are not allowed.

FIRST AID
For any spilled acids or bases, first aid should begin by washing with lots of water.
ALWAYS USE WATER FIRST.
1. For base burns, follow the water wash by rinsing the affected area with a 5%
ammonium chloride, then wash it again with water.
2. For acid burns, follow the water wash by rinsing the affected area with a sodium
bicarbonate solution, then wash it again with water.
3. For heat burns, apply a thick paste of sodium bicarbonate and water or burn
ointment. If third degree skin burned happened, do not apply paste or grease,
see a doctor immediately.
4. For minor cuts, wash it with soap and water, then apply it with a 10% betadine.
Apply an antiseptic ointment and a clean bandage.
5. In cases of serious burns, first aid should be immediately administered and
competent medical help should be sought as soon as the first aid treatment has
been applied.
6. Speed is essential in the treatment of chemical burns, particularly when the eyes
are involved.

CHEMISTRY DEPARTMENT SAFETY RULES

1. Do not work in the laboratory unless your instructor is present to supervise your
work. A qualified person must be present to see that only safe procedures are
followed, also this person could provide immediate aid in case of an accident.
2. Do not carry out any unauthorized experiment. Perform only those
experimental steps in the printed manual, or those given directly to you by your
instructor.
3. Do not work under any condition that you believe to be unsafe to you or to
others. If such condition exists (overcrowded area, unsafe actions by another
student), report it immediately to your instructor or to a faculty member in
charge.
4. Wear approved eye protection at all times in the laboratory. Approved eye
protection means wearing safety goggles. Eyes are very susceptible to chemical
injury and must be fully protected all the time. Be aware that even when you are
not working, a person nearby may be carrying out a chemical procedure that
might affect you, thus; wearing eye protection is a must at all times.
5. Contact lenses must not be worn in the laboratory. All types of contact lenses
may trap a chemical against the eye tissue and cause permanent eye damage.
6. Do not work with a chemical above or near your face. Holding a beaker up to
look at what is in the bottom, or filling a burette, which is higher than eye-level,
can result in a splash down onto your face.
7. Many chemicals are toxic/or corrosive. Do not assume that any chemical reagent
is safe and that it does not require careful handling.
8. Do not taste or ingest any chemical in the laboratory. It may be toxic. Even NaCl
may be contaminated and be unsafe. For the same reason, you cannot bring food
or drink inside the laboratory, or eat in the laboratory.
9. Never pipette by mouth. Drawing up a liquid into a pipette should be done only
with a rubber bulb or water aspirator.
10. Never pipette directly from a reagent bottle. Transfer only necessary amount of
liquid reagents to a secondary container, such as a clean dry beaker.
11. Avoid skin contact with any chemical. Keep the outside of reagent containers, all
of your equipment and your desktop free from chemical spills. Wear gloves if
instructed to do so.
12. Do not inhale reagent fumes. Odor tests are to be made only when specifically
directed to do so. Use a waving motion of your hand to bring the vapor near
your nose.
13. Fume hoods must be used whenever toxic or corrosive vapors are released
during the work you are doing. Use the hood when directed to do so. If fumes
develop unexpectedly, cover the container and take it to the hood at once. Work
with concentrated hydrochloric acid, nitric acid, or acetic acid or with bromine,
chloride, or hydrogen sulfide only in the fume hood.
14. Alkalis are particularly corrosive. Contact with NaOH and other alkaline
chemicals must be avoided. Strong bases must be handled with great caution
because they attack tissues so rapidly.
15. Do not heat a test tube containing a liquid over an open flame or directly on a hot
plate. To heat a test tube, hold it in a beaker of hot water. Liquids heated over
an open flame may erupt violently and splash onto you directly.
16. Do not add water to a concentrated reagent, especially concentrated sulfuric acid.
Keep the mixture as dilute as possible; add the reagent to water. Addition of
concentrated sulfuric acid to water causes much heat formation and may result
in spattering of this corrosive reagent.
17. Handle liquid reagent with care. When pouring a liquid, grasp each container so
that drips cannot contact your fingers. When using a polyethylene bottle, do not
pour from it or squeeze it in any manner that might result in a stream of liquid
getting to you, or to someone nearby.
18. Dry all wet glassware before storing it in your locker. Keep a cloth towel in your
locker to use for drying glassware and wiping your hands.
19. Carry glass tubing and glass thermometers only in an upright position. On
impact, glass tubing can snap and become a dagger.
20. To insert glass tubing or a thermometer into a rubber stopper, fire polish the
ends of the tubing first, lubricate the stopper hole with water or glycerin, then
insert the tubing cautiously, using a towel or rag to protect your hands. If
handled improperly, glass tubing can break and become razor sharp when
inserted into a stopper.
21. All broken glass laboratory waste must be placed into the special glass disposal
boxes at the end aisles in each laboratory room. Only paper products go
into the regular trashcans.
22. Waste “sharps objects” such as syringes, syringe needles, razor blades and
scalpels must be placed in the special disposal bottles provided in the laboratory.
23. If a chemical splashes into your eyes, get help immediately. If someone gets a
chemical in her/his eyes, you should ask for help from the instructor
immediately. Wash the eye/s thoroughly with a stream of water. Hold the
eyelids open.
24. Any chemical that comes in contact with your skin should be washed off with
water immediately.
25. Know the location of fire extinguishers, fire blankets, and safety showers, in case
of fire. Keep acetone and any other organic liquid at least ten (10) feet from an
open flame.
26. Proceed cautiously when handling hot objects. Use a towel as a hot pad when
handling hot objects. Hot glass looks like cold glass. In case of burn, immerse in
water immediately. Notify your instructor. Apply clean moist cloth or bandage.
Seek medical attention.
27. Know the evacuation sirens and exit route from your laboratory. When the fire
alarm sounds, stop what you are doing and immediately exit from the
laboratory. Go down the stairs and immediately exit from the building. Wait
outside for instructions.
28. Immediately report any accident to your instructor no matter how minor it may
seem to you. A professional medical person should treat cuts, burns, chemical
burns, and inhalation or ingestion of chemicals as soon as possible. Neither
students nor chemistry staffs are qualified to make medical decisions.
29. Only neutral aqueous solutions go down the sink drain. Waste determination
and disposal are done by the faculty and or the staff. Check with your instructor
before disposing off any chemical. All chemical/wastes are to be sorted into the
appropriate waste container and the identity and amount must be logged in the
accompanying inventory sheet. Check with your instructor for specific details.
30. Clean your workbench with a damp sponge. Neutralize all acid spills with
sodium bicarbonate and wash with a wet sponge. Shut gas jets completely.
Wash your hands. Leave the area safe for the next person.
31. Do not take any chemical out of the laboratory for any reason. It is illegal! You
may be liable.
 COLORIMETRY

Colorimetry is the ability of colored substances to absorb a specific amount of

light energy at a given wavelength of light. The amount of absorption is directly related
to the amount of substance.

BEER-LAMBERT’S law states that the absorption of light energy at any

wavelength is solely dependent on the concentration of the absorbing material and the
length of the light path through the absorbing medium. It is expressed as:

Absorbance = log10 lo
lt
Where:
l0 = ratio of incidence light
lt = ratio of transmitted light
 ELEMENTS OF SPECTROPHOTOMETRY

Concentration of dissolved material can be quantitatively determined by

measurement of light absorption. Spectrophotometry refers to measurements made
with UV, visible and infra-red light, while colorimetry refers only to measurement with
visible light. A spectrophotometer is a combination of a spectrometer and a
photometer. Spectrometer is a device used for producing light of any selected color. It
is called a monochromator when used as a part of spectrophotometer. It is calibrated to
express the color of the monochromatic light and expressed in terms of wavelength.
Photometer is a device used to measure the intensity of light. With spectrometer, it is
used to measure the intensity of the monochromatic beam produced by the
monochromator.

Spectrophotometric measurements are easy to perform. The solution under

examination is placed in a calibrated test tube or cuvet, the proper wavelength is
selected and the cuvet then placed in the light plan so that this wavelength passes
through the solution and it’s container. Any transmitted light is directed onto a
photosensitive device which converts the radiant energy to electrical energy. The
electrical current generated, in turn, can be measured by a meter.

PARTS OF A SPECTROPHOTOMETER

1. Sample control
2. Scale
3. Signal lamp
4. Light control
5. Wavelength control
6. Wavelength scale
7. Zero control

Operating Procedure for Spectronic 20

1. Turn the power switch knob clockwise. Allow the instrument to warm up for
five (5) minutes.
2. Select the desired wavelength, by turning the wavelength control knob to the
desired setting as indicated on the wavelength scale.
3. Adjust the zero control knob so that the meter needle reads zero in the
transmittance scale.
 4. Insert into the sample holder the cuvette containing distilled water or some other
reference solution.
5. Adjust the light control knob until the meter reads 100% transmittance or zero in
the absorbance scale.
6. Remove the reference liquid from the cuvette and replace it with a tube
containing the sample or reference solution.
7. Read directly the % T or A at the prescribed wavelength.

CHROMATOGRAPHY

Chromatography is a process wherein the solution of substances to be separated

is passed through a region of immobilized substance through various means and allows
for the differential migration of the components of the solution. It is composed of two
(2) phases namely the mobile phase and the stationery phase. The mobile phase
contains the mixtures to be separated while the stationery phase contains solid or
liquid. Separation depends on the differences in the extent to which individual
components in the moving phase interact with the stationery phase. This differential
interactions result in the migration of the components in a mixture.

Different Types of Chromatographic Techniques

1. Paper Chromatography – this is a special type of filter paper that is used

immobile support for a liquid stationery phase. It is used for the separation of
amino acids, sugars, oligosaccharides, fatty acids, alkaloids and other natural
products.
2. Column Chromatography – this is a glass column that is filled with a solid
stationery phase such as silica gel, calcium phosphate gel, gel, sucrose, calcium
carbonate, and alumina. The solid phase interacts with the liquid moving phase
containing the sample by adsorption. The sample to be separated binds at the
surface of the solid particles.
3. Gel Filtration Chromatography – it is composed of cross-linked dextran, which
are tiny gel particles, this serves as the solid stationary phase and molecular
sleves. When placed in water, having a porous network, the particles absorb
water and swell into larger granules. When samples in the moving phase enter
the stationery phase, they interact with the gel granules. Smaller molecules
enter the pores and appear last in the eluate. Larger molecules do not penetrate
but move on and dissolve in the moving phase through the column. It is used in
the separation of protein mixture, and molecules with different molecular
weights.
 4. Ion-Exchange Chromatography – it is an adsorption chromatography which is
composed of the adsorbed material and the adsorbent. The adsorbent is an ion-
exchanger, which is an insoluble matrix to which charged groups are covalently
bound. There are two types of ion-exchanger namely: cation exchanger and the
anion exchanger. Ion-exchange chromatography is used mostly in the
separation of protein molecules with the formation of multiple bonds between
the charged groups on the protein and the available groups of opposite charge
on the matrix.

ELECTROPHORESIS

Electrophoresis is the movement of a charge particle in an electric field.

Movement of substances occurs in a liquid medium mostly buffers which is supported
by an inert solid substances such as paper or solid gel substance. Charge molecules
move either to the cathode or to the anode if electricity is applied. Ions mobility
depends on the net charge of the molecule, radius of the molecule and the viscosity of
the liquid medium and the applied voltage.

Different Types of Electrophoresis

1. Disc Gel Electrophoresis – the polyacrylamide gel is placed in a small tube

supported in a vertical position, serves as a support for the liquid buffer. Substances
migrate through the tube column as a sharp, narrow zones. Gel movement is based
partly on electrophoretic mobility and molecular weights. It is used in the
determination of enzymes, nucleic acids, and other classes of compounds.
2. Paper Electrophoresis – paper strip is used as a support medium. Two half-cells
containing buffer solutions are connected to the paper and are being wet with the
buffers. Sample solution is applied midway between the two (2) half cells and
current with a high voltage is applied through the system. Separating substances
migrate towards the electrodes depending on their charge.
3. Starch Gel Electrophoresis – starch gel is used as the supporting medium. Starch
is evenly layered over a template and is allowed to set at room temperature and
refrigerated for at least two (2) hours before use. Filter paper strip is used to contain
the samples and placed in between the two (2) gel portions over the template, then
placed on top of the buffer trays containing the electrode buffer. Power supply is
connected to terminals of the buffer tray, and electric power is set at a constant
voltage and electrophoresis is run for 16-20 hours.
 TABLE CENTRIFUGE

Centrifuge is one of the most frequently used apparatus in the laboratory. It is a

potentially dangerous machine. Proper care is needed to prolong the life of this
machine. Thus safety of the user must be monitored.

Procedure for Use:

1. Only similar test tubes can be used as containers.

2. Exactly balance the contents of sample tubes against another. Unequal weights
may result in breakage of the tubes, loss of sample and damage to the machine.
3. Avoid spilling of samples, and if it does occur, make sure all are wiped off.
4. Plug the machine to an electrical supply, set the timer and the dial to the
required speed and time.
5. Wait for the machine to stop by itself. Never stop the centrifuge by manually
controlling the rotor.
6. Remove samples from the tubes.
7. After using the machine, thoroughly dry or wipe all spillage before keeping.

pH METER

The pH meter is a direct-reading instrument for making highly precise pH and

potential measurements. It indicates the hydrogen ion-concentration in a test solution
by responding to the potential developed by an electrical cell. The cell consists of two
electrodes namely the glass and the reference electrodes.

Glass Electrode – is a thin membrane electrode glass, which is bath with fixed
solution of constant pH, which in turn bathes a metal having a constant potential with
respect to their solution. When the electrode is immersed in the solution, a potential
difference develops, the magnitude of which depends on the hydrogen ion
concentration of the solution. Potential differences are then measured by a
potentiometer.

Reference Electrode – consists of an internal element (silver-silver chloride) and

an outer glass body, which is filled with an electrolyte solution (KCI). This establishes a
constant potential at the internal element and acts as an electrolyte conducting bridge
between the element and the test solution. This electrical connection is maintained by a
flow of electrical potential at the liquid junction.
 In the measurement of pH, the electrodes are first immersed in a buffer solution
of known pH. The zero point is adjusted by turning the asymmetry control until the pH
reading coincides with the pH of the buffer. The standardization automatically
compensates for the various potentials in the electrode system. Subsequent immersion
of the electrodes in a test solution causes a potential difference proportional to the pH of
the solution. This potential registers directly as pH on the standard or expanded scale
of the pH meter.

Procedure for Use:

1. Set the temperature control knob at about 25.

2. Immerse the electrodes in a standard buffer solution.
3. Depress the standard scale selector.
4. Depress the pH selector and take the reading. If the pH registered on the scale is
not the same as the pH of the standard buffer adjust the pH reading using the
asymmetry control knob.
5. Depress the standby button.
6. Rinse the electrodes with distilled water and dry gently using tissue paper. DO
NOT HOLD the electrodes with your fingers or apply pressure.
7. Immerse the electrodes in the solution of unknown pH, depress the pH selector
and take the reading on the scale. This is the pH of the unknown solution.
8. Immerse the electrodes in distilled water and leave the instrument at standby
position.

TITRATION
 Titration – is a process of determining the quantity of given constituent present
in a compound using a standard solution that interacts with or neutralizes the
constituent. Acids and bases neutralize each other. Therefore, a known concentration
of a base solution can be used to titrate an unknown concentration of acid solution.

TYPES OF TITRATION METHOD:

1. Direct Method – when a standard acid solution is added to an alkali solution

until an endpoint is reached.
2. Residual or Indirect Method – when an excess of the standard acid solution is
added to the unknown alkali solution and the excess acid titrated with standard
alkali solution.

Components of Titration:

1. Titrant –

2. Analyte –
Name_____________________________________________DatePerformed________
Course/Year/Section_______________________________Rating_______________
Exercise No._____
Typical Animal Cell
1. Draw a typical animal cell, label the parts and give their functions.
Name_____________________________________________DatePerformed________
Course/Year/Section_______________________________ Rating_______________
Exercise No._____
Typical Plant Cell
1. Draw a typical plant cell, label the parts and give their functions.

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
Cell Macromolecules

The cell is composed of organelles with their corresponding macromolecules.

These organelles vary in density and thus can be separated by differential
centrifugation, and their chemical composition determined using common qualitative
tests.
Preparation of Cell Homogenates:
Wash 5 grams of chicken liver with 0.25M sucrose solution. Cut into fine pieces, then
transfer into a mortar and add 20 ml of 0.25M sucrose solution and macerate it. Subject
the mixture to differential centrifugation.

1. Centrifuge the homogenized cells in sucrose solution in a test tube at 66 x g for 10

minutes. Transfer the centrifugate in another test tube. Discard the residue
2. To the centrifugate, centrifuge at 8,000 – 10,000 x g for 10 minutes. Transfer the
centrifugate in another test tube and label the test tube with the residue as Test
Tube 1.
3. To the centrifugate, centrifuge at 15,000 x g for 20 minutes. Transfer the
centrifugate in another test tube and label the test tube with residue as Test Tube
2.
4. Label the centrifugate on the above procedure as Test Tube 3.
5. Add 2 ml of 0.25M sucrose solution to Test Tube 1, 2 and 3 and divide each into
four equal portions and perform the following qualitative test.

Test for Protein:

Reagent:
10% NaOH, 0.5% CuSO4

Procedure:
1. To the first portion, add 5 drops of 10% NaOH and 5 drops of 0.5% CuSO 4.
Shake. Observe.

Result:
Test Tube 1

Test Tube 2

Test Tube 3

Test for Lipids

Reagent:
Sudan IV Crystals
Procedures:
1. To the second portion, add a pinch of Sudan IV crystals. Shake very well.
Observe.
Result:

Test Tube 1

Test Tube 2
 Test Tube 3

Test for Nucleic Acids

DNA (Deoxy Ribonuleic Acid)
Reagents:
Diphenylamine reagent, concentrated H2SO4
Procedure:
1. To the third portion, add 5 drops of diphenylamine and 2 drops of
concentrated H2SO4.
2. Heat in a water bath for 5 minutes. Observe.
Results: heating DNA with diphenyl amine in acid solution produces a blue
color (Dishe reaction)

Result:
Test Tube 1

Test Tube 2

Test Tube 3

RNA (Ribonucleic Acid)

Reagents:
Orcinol reagent
Procedure:
1. To the fourth portion, add 5 drops of Orcinol reagent.
2. Heat in a water bath for 5 minutes. Observe.
Results: orcinol reagent is used to determine the presence of pentoses and
nucleotides that contain pentose sugars. When pentoses are treated with
orcinol, furfurals are formed and will yield a blue-green compound in the
presence of ferric ions. This is also known as Bial’s test.

Data and Results:

Test Tube Number Nucleic Acid
DNA – RNA Protein Lipids
Nuclear Fraction

Mitochondrial Fraction

Lysosomal and
Ribosomal Fraction
In tabulated form, state the different cell organelles, their chemical composition, and
their functions.

Cell Organelles Chemical Composition Function

Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
Translocation of Materials

Constant exchange of material takes place between the cell and its external
environment. This is accomplishing through diffusion, carrier-mediated transport, or
bulk transport.

Translocation through an Artificial Membrane

A. Osmosis

Materials:
thistle tube, beaker, rubber band, iron stand, burette clamp, yema
wrapper, table salt, table sugar
Procedure:
1. Fill the bulb of a thistle tube with saturated salt solution up to its constricted
portion.
2. Cover the thistle tube with yema wrapper and secure it with a rubber band.
3. Immerse the bulb of the thistle tube in a big beaker with water suspending it
by means of a burette clamp to an iron stand.
4. Be sure that the levels of both liquids are the same.
5. Observed the level of the solution inside the thistle tube after an hour.
6. Repeat the same procedure using sugar solution.
7. Observe.

Result:
B. Dialysis

Materials:
Yema wrapper, rubber band, starch paste, iodine, glucose, Benedict’s
solution

Procedure:
1. Place 10 ml of starch paste with 10 drops of glucose solution in a yema wrapper
and tie with rubber band.
2. Immerse this in a beaker with 150 ml of water and five (5) drops of iodine
solution for ten (10) minutes.
3. Observe the change in color inside the yema wrapper.
4. Place 2 ml of the water from the beaker in a test tube.
5. Add 10 drops of Benedict’s solution and heat the solution in a water bath for 5
minutes.
6. Observe.
Result:

Translocation through the Cell Membrane

 A. Animal Cell

Materials:
Citrated blood, test tube, test tube rack, glass slide, microscope, black thread,
medicine dropper, isotonic, hypotonic and hypertonic solutions

Procedure:
1. Place 1 ml of isotonic, hypotonic, and hypertonic solutions in each of 3 test tubes.
2. Place these test tubes on a test tube rack and stretch a black thread across the
back of the test tube rack.
3. Add two (2) drops of blood in each test tube and determined the time for
hemolysis to take place. This is indicated by the black thread seen through the
solution.

Result:

Isotonic Solution Hypertonic Solution Hypotonic Solution

Observation:

Record the time of appearance of the black thread at the back of the test tubes:

Test tube Time

1. _____
2. _____
3. _____
Place a drop of sample from each test tube in a glass slide and cover. Observe under the
microscope. Draw the blood cell.

Plant Cell
Materials:
Leaf of rheo discolor, glass slide, salt, water, microscope
Procedure:
1. Place a thin piece of the leaf on a slide.
2. Add small amount of water.
3. Draw the cell seen under the microscope.
4. Place a pinch of salt on one side of the specimen.
5. Observe and draw the cell seen under the microscope.

C. Diffusion
Materials:
Beaker, KMnO4 crystals, intermediate pad

Procedure:
1. Place 20 ml water into a beaker.
2. Place the beaker over a piece of intermediate pad.
3. Get the temperature of the water in the beaker.
4. Allow the water inside the beaker to become very still.
5. Carefully place several crystals of KMnO 4 at the bottom of the beaker where the
lines are perpendicular to the beakers edge.
6. Note the time required for the colored ions to travel 1 cm along a line toward the
center.
7. Observe.
8. Repeat the procedure by heating the water inside the beaker to 800C.

Observation:

a. At room temperature

b. At 800C

D. Surface Tension
Materials:
Margarine, watch glass, bile solution, 0.5% Na2CO3
Procedure:
1. Apply thin coating of margarine to the bottom of two watch glasses.
2. Cover the margarine coating with bile solution on the 1 st watch glass and 0.5%
Na2CO3 on the other.
3. Allow the solution to stand for 30 minutes.
4. Pour off the solutions and rinse each watch glass with running water.
5. Hold the watch glass up to the light and account for the difference at the
margarine coating.
6. Observe.

Questions:
1) Define the following terms:
A. Osmosis –

B. Dialysis –

C. Isotonic solution –

D. Hypertonic solution –

E. Hypotonic solution –

F. Hemolysis –

G. Crenation –

H. Plasmolysis –

2) If a plant is placed in salt water, what will happen to it? Explain.

3) In an animal tissue, why is concentrated salt or sugar solution considered

as a preservative?
Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
pH and BUFFERS

Biological processes are greatly affected by the presence of hydrogen or hydroxyl

ion, in the medium in which they occur. The kidney is an organ involved in this
homeostatic mechanism of the body to maintain pH. pH is a way of expressing the
acidity and basicity of a solution. It is defined as the logarithm of the reciprocal of the
hydrogen ion concentration (H+) or the negative logarithm of the hydrogen
concentration: Thus,

pH = log 1 or pH = - log (H+)

(H+)
Tissues produce acids during metabolism and these acids are neutralized by buffer
systems to maintain normal acid-base balance. Maintenance of an optimum pH in the
environment of plant and animals cells is vital to the life of an organism. The function
of butter is to control pH, which is very essential for enzymes-catalyzed reactions
within the cell. Buffer is a mixture of a week acid and its conjugate base or a weak
base and its conjugate acid, expressed as:

HA ----- H+ + A

Ka = (H+) (A-)
HA

Where:
Ka = dissociation constant of the weak acid
H+ = hydrogen ion concentration
HA = concentration of the acid

From this relationship Henderson-Hasselbach equation can be derived:

pH = pKa + log A
HA

A. Measurement of pH

Reagents:
Different fruit-juices (natural), vegetables (grinded or squeezed)

Materials:
Hydrion pH paper, pH meter, litmus paper

Procedure:
1. Prepare 5 ml. of each of the following samples:
A. Aspirin tablet
B. Baking soda (5% solution)
C. Defibrinated blood
D. Fresh milk
E. Freshly voided urine
F. Saliva
2. Determine the pH using hydrion pH paper by dipping the pH paper in the
sample for 10 seconds. Match the color produced in the pH paper with the color
chart to determine the pH of the sample.
3. Check the pH obtained by using the pH meter.
4. Compare the pH readings of the two.
5. Write your result in a tabulated form.
B. Preparation of Buffers:
Reagents:
0.1 M CH3COOH, 0.1 M NaCH3COO, 0.1 M HCI, 0.1 H3PO4
0.1 M NaH2PO4, 0.1 M NaHPO4, 0.1 M NaOH

Material:
Graduated cylinder, weighing balance, spatula, beaker, stirring rod, pH
meter

Procedure:
1. Calculate the volume ratios by which the following solutions should be mixed to
produce buffer solutions.

a. 0.1 M CH3COOH and 0.1 M NaCH3COO, pKa = 4.73

b. 0.15 M H3PO4 acid, 0.15 M NaH2PO4 and 0.15 M Na2HPO4, pKa = 7.2

2. Show your calculations and have it checked by your instructor before preparing
the solution.
3. Measure the required volumes of the reagents needed and proceed with the
buffer preparations.
4. Check the pH of your buffer with the standardized pH meter.
5. If the measured pH of the buffer deviates from the assigned value, add dropwise
of either 0.1 M HCI or 0.1 M NaOH to bring the buffer to the desired pH value.
6. Record your results. Keep the prepared buffer solutions in the refrigerator until
needed.

B. Buffering Action of Blood Serum:

Reagents:
0.1 M NaOH, 0.1 M HCI, phenolphthalein, methyl orange, blood serum

Materials:
Test tubes, test tube rack, pipette, medicine dropper

Procedure:
1. Prepare four (4) clean test tubes and number them one (1) to four(4).
2. Add 5 ml of water to test tube 1 and 2 and 4 ml of water to test tubes 3 and 4.
3. Add 1 drop of 0.1 M NaOH to test tube 1.
4. Add 1 drop of 0.1 M HCI to test tube 2.
5. Add 1 drop of phenolphthalein each to test tubes 1 and 3.
6. Add 1 drop of methyl orange each to test tubes 2 and 4.
7. Add 2 drops of blood to both test tubes 3 and 4.
8. To test tube 3, add 0.1 M NaOH dropwise until it matches the color of test tube 1.
Record the number of drops of 0.1 M NaOH used.
9. To test tube 4, add 0.1 M HCI dropwise until it matches the color of test tube 2.
Record the number of drops of 0.1 M HCl used.
10. Record your results in a tabulated form.

Questions:

1. Show calculations on the preparation of buffers. Write their correct chemical

formula. Use the Henderson-Hasselbach equation for your computation.

2. How would you account for the buffering action of blood serum?

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
Carbohydrates

Carbohydrates are organic substances composed of certain chemical groups, such as

alcohols, aldehydes, or ketone groups. These compounds include natural sugar which
are largely distributed in nature, and are classified as monosaccharides, disaccharides
or polysaccharides based on the complexity of their molecules.
General Procedure:
Prepare seven (7) test tubes with 10 drops of each of the following carbohydrates:
TEST TUBE CARBOHYDRATES
1 Glucose
2 Galactose
3 Fructose
4 Maltose
5 Lactose
6 Sucrose
7 Starch
Make sure that these sample solutions are freshly prepared.

General Test for Carbohydrates:

1. Molisch Test:
Reagents:
Molisch Reagent, concentrated sulfuric acid

Procedure:

1. Add 5 drops of Molisch reagent to all 7 test tubes and shake.

2. Add 10 drops of concentrated sulfuric acid, pouring the acid carefully down the
side of the test tube in a slant position.
3. Observe the color formed at the junction of the two layers.

Observation:
 2. Test for Reducing Sugars:
1. Benedict’s Test:
Reagent:
Benedict’s reagent

Procedure:
1. Add 5 drops of Benedict’s reagent to all 7 test tubes.
2. Heat in a water bath for 5 minutes.
3. Observe for the change in color.
Observation:

2. Nylander’s Test:
Reagent:
Nylander’s reagent

Procedure:
1. Add 5 drops of Nylander’s reagent to all 7 test tubes.
2. Heat in a water bath for 5 minutes.
3. Observe for the change in color.
Observation:

3. Osazone Crystals Formation :

Reagent:
Phenyl hydrazine solution

Procedure:
1. Add 5 drops of phenyl hydrazine reagent to all 7 test tubes.
2. Heat in a water bath for 5 minutes.
3. Observe for the formation of crystals.
Observation:

4. Tollen’s Test:
Reagent:
Tollen’s Reagent
Procedure:
1. Add 5 drops of freshly prepared Tollen’s reagent to all 7 test tubes.
2. Warm in a water bath for 5 minutes.
3. Allow to stand for another 5 minutes.
4. Observe for the change in color.
Result:

5. Barfoed’s Test:
Reagent:
Barfoed’s reagent

Procedure:
4. Add 5 drops of Barfoed’s reagent to all 7 test tubes.
5. Heat in a water bath for 5 minutes.
6. Note the time required for the solution to give a definite color.
Observation:
6. Seliwanoff’s Test:
Procedure:
1. Add 5 drops of Seliwanoff’s reagent to all 7 test tubes.
2. Heat in a water bath for 5 minutes.
3. Observe.
Observation:

Test for Polysaccharide:

1. Iodine Test:
Reagent:
Iodine Solution

Procedure:

1. Add 5 drops of Iodine solution to all 7 test tubes.

2. Shake well.
3. Observe for the change of color.
Result:

Questions:
a. What is the general test used for carbohydrates?
b. What is the linkage that binds one monosaccharide to another?

c. Give the importance of all test performed on carbohydrates.

a. Molisch test –

b. Benedict’s test –

c. Nylander’s test –

d. Barfoed’s test –

e. Seliwanoff’s test –

f. Formation of osazones –

g. Iodine test –

Name: Date Performed:

Course/Year/Section: Rating:
 Experiment No.
Lipids

Lipids are water – insoluble organic compounds that can be extracted from cell and
tissues by non-polar solvents like ether, chloroform, benzene, etc. Lipids that contain
fatty acid are saponifiable, while those without fatty acid like steroids, are non-
saponifiable.
General Procedure:
Prepare 6 test tubes and add 10 drops each of the following tests for lipids
Test Tube Test Solution
1 coconut oil
2 corn oil
3 margarine
4 oleic acid
5 beef tallow
6 pork fats

1. PHYSICAL PROPERTIES
A. Solubility Test
Reagents: chloroform, alcohol, 5% NaOH, 5%HCI, water, ether,

Procedure:
1. Add 1 ml each of the following on all 6 test tubes. Shake well. Observe
A. Chloroform
B. Alcohol
C. Water
D. NaOH
E. HCI
F. Ether
2. Write your results in a tabulated form.

B. Other Physical Properties:

Reagents: coconut oil, butter, margarine, oleic acid, lanolin, beef tallow.
Procedure:
1. Observe and record the appearance, consistency, color and odor of each of the
lipid samples.
2. Write your answers in tabulated form.

C. Spotting Effect:
Reagents: alcohol, ether, coconut oil

Procedure:
1. Place a drop of coconut oil in a piece of filter paper.
2. On the same spot, place a drop of alcohol. Observe.
3. Repeat the procedure using ether.
Result:
A. oil and alcohol

B. oil and ether

2. CHEMICAL PROPERTIES:
A. Emulsification of Fats and Oils:
Reagents: coconut oil, corn oil, margarine, oleic acid, pork fats, beef tallow.

Procedure:
1. Place three (3) drops of coconut oil into two (2) dry test tubes.
2. Add 3 ml of hot water to each test tube.
3. In the first tube, add 1 ml of soap solution.
4. Shake each test tube vigorously, then let stand and observe.

Result:
Coconut oil: Test Tube 1

Test Tube 2

Corn oil: Test Tube 1

 Test Tube 2

Oleic acid: Test Tube 1

Test Tube 2

Margarine: Test Tube 1

Test Tube 2

Beef Tallow: Test Tube 1

Test Tube 2

Pork Fats: Test Tube 1

Test Tube 2

B. Test for Unsaturation:

Reagents: coconut oil, corn oil, oleic acid, margarine, beef tallow, pork fats, CCI 4, Br2
solution.

Procedure:
1. Test the substance listed below to determine the number of drops of Br 2 solution
(w/ CCI4 as the solvent) that can be decolorized. The first faint persistence of the
color bromine is the end-point of the reaction. Dissolve approximately 1 ml of each
substance to be tested in 5 ml of CCI4 before adding the bromine solution.
2. Enter the name of the substance tested as well as the amount (drops of bromine
solution) required.

Substance Tested Drops of Bromine solution absorbed

Coconut oil
Corn oil
Oleic acid
Margarine

C. Acrolein Test:
Reagents: KHSO4 crystals, glycerol

Procedure:
1. Place about one (1) gram of KHSO4 in a dry test tube.
2. Add 10 drops of glycerol.
3. Heat slowly at firs to prevent evolution of SO2.
4. Then heat vigorously and note the odor of acrolein.
5. Repeat the procedure using the other lipid sample and compare the result with
glycerol.
6. Write your result in a tabulated form.

D. Hydrolysis of Fat by Dilute Acids:

Reagents: 10% HCI

Procedure:
1. Place one (1) ml of coconut oil in a test tube.
2. Add two (2) ml of 10%HCI.
3. Place in a water bath for 15 minutes.
4. Pour contents in an evaporating dish and allow to cool at room temperature.
5. Test with blue litmus paper.
6. Identify the fatty acid present.
7. Repeat the procedure using the other lipid samples.
8. Write and record your observations in tabulated form.

Result:

E. Test for Cholesterol:

Reagents: fatty acids extract from Procedure D, chloroform, conc. sulfuric acid
Procedure:
1. Place a few crystal of cholesterol in a clean dry test tube.
2. Add 2 ml. of chloroform and 5 drops of concentrated sulfuric acid.
3. Observe for the change in color of the solution from bluish red to purple.
4. Repeat the procedure using the fatty acid extract from Procedure D.
5. Observe.

Questions
1) Butter can become rancid as a result of hydrolysis by microorganisms.
Which of the fatty acids are responsible for the bad odor associated with rancidity?

2) Predict the solubility of coconut oil in water, ethanol, and ether.

3) What is the function of soap in dishwashing or washing greasy hands?

4) What characteristics of soap make it a good emulsifying agent?

5) Give three (3) examples of lipid that are positive in acrolein test. What
cause the positive reaction?

6) What are the products of hydrolysis of fats?

7) What are the conditions and compounds necessary to hydrolyze fats?

Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
Nucleic Acids

Nucleic acids are complex organic compounds composed of nucleotides. Each

nucleotide is composed of a pentose sugar, a nitrogen base, and phosphate. Depending
on the pentose sugar present, two kinds of nucleic acids are known: DNA whose sugar
is deoxyribose, and RNA with ribose sugar. Qualitative analyses of nucleic acids based
on the presence of phosphorus as phosphate, pentose sugar, and the nitrogen which are
derivatives of purine and pyrimidine.

Isolation of RNA from Yeast

Place 125 ml of water in a beaker. Add 25 ml of 1% NaOH and 20 grams of dried yeast.
Heat the mixture in a water bath for 30 minutes, constantly stirring it while heating.
Remove the mixture from the water bath and filter it with a cheesecloth. Cool the
filtrate and add 3 drops of acetic acid. Evaporate the filtrate up to 50 ml. Cool and pour
with vigorous stirring into 100 ml of 95% ethanol with 1 ml of concentrated HCI to
precipitate the RNA. When RNA has settled, decant, dry the RNA in a petri dish at
room temperature.

Solubility Test
Reagents: Alcohol, dilute HCI, dilute NaOH

Procedures:
1. Place in a separate test tube 1 ml each of the following: cold water, hot water,
alcohol, dilute HCI, and dilute NaOH.
2. Add a pinch of the yeast RNA to each test tube. Shake well. Observe.

Result:

Cold water

Hot water

Alcohol

Dilute HCI

Dilute NaOH

Hydrolysis of RNA in Acid

Reagents: 10% H2SO4

Procedure:
1. Place a small amount of the yeast RNA in a test tube.
2. Add 10 ml of 10% H2SO4, cover, and boil in a water bath for 30 minutes.
3. Divide the contents of the test tube into 4 equal portions. Label the test tubes 1,
2, 3 and 4. Perform the following tests below.

1. Benedict’s Test
Reagents: Solid Na2CO3, Benedict’s solution

Procedure:
1. Neutralize the solution in test tube 1 with solid Na 2CO3, let stand for 2 minutes
and then decant.
2. Add 1 ml of Benedict’s solution in the test tube and place in a water bath for 5
minutes. Observe.

Result:

2. Test for Purine

Reagents: NH4OH 1%, AgNO3

Procedure:
1. Treat the solution in test tube 2 with 3 drops of ammonia water to make it
alkaline.
2. Add five drops of 1% AgNO3 solution and let stand for five minutes. Observe.

Result:

3. Test for Pentose

Reagent: Bial’s reagent

Procedure:
1. Add the solution in test tube 3, add 5 ml of Bial’s reagent.
2. Heat test tube in a boiling water bath for 10 minutes. Observe.

Result:
 4. Test for Phosphate
Reagent: Ammonia water, 6N HNO3, (NH4)2 MoO4 solution

Procedures:
1. In test tube 4, and add five (5) drops of ammonia water, acidify with 2 drops of
6N HNO3.
2. Add 1 ml of (NH4)2MoO4 solution.
3. Heat the mixture in a water bath for 5 minutes. Observe.

Result:

Repeat these tests using unhydrolyzed RNA and compare the result by completing the
table:

Hydrolyzed RNA Unhydrolyzed RNA

Benedict’s Test
Test for Purine
Test for Pentose
Test for Phosphate

Questions:

1. Give the role of the following reagents in the isolation of RNA:

A. 1% NaOH –

B. Acetic Acid –

C. 95% ethanol –

2. Give the role of 10% H2SO4 in the hydrolysis of RNA.

3. State the biologic function of the following:
a. DNA –

b. mRNA –

c. tRNA –

d. rRNA –

4. What are the products of the hydrolysis of nucleic acids?

5. Is DNA hydrolyzed by alkali? Explain.

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
Proteins

Proteins are nitrogen containing organic compounds composed of amino acid

connected to one another by peptide bonds. Each amino acid has a basic amino (NH 2)
group and an acidic carboxyl (COOH) group, with a characteristic radical group. The
chemical properties of amino acids are due to these group, and the chemical properties
of the group of the proteins formed from these amino acids, in turn are dependent on
their chemical properties.

NOTE: Use 2% albumin solution in all these experiments.

Coagulation Tests

1. By alcohol
Reagents:
acetic acid, 95% ethanol

Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add 10 drops of 1% acetic acid and 10 drops of 95% ethanol.
3. Shake the mixture.
4. Observe.

Result:

2. By heat
Reagent:
acetic acid

Procedure:
1. Place 1 ml of egg albumin solution in a test tube and heat to boiling.
2. Observe.
3. Add two (2) drops of acetic acid in the test tube and note the effect.

Result:

Precipitation Tests

1. By salts of heavy metals

Reagent:
5% ferric acid

Procedure:
1. Place 1 ml of egg albumin solution in a test tube.
2. Add 5 drops of 5% ferric chloride solution. Shake the mixture.
3. Note the change in color of the solution.

Result:
2. By strong mineral acids
Reagents:
concentrated nitric acid, concentrated sulfuric acid

Procedure:
1. Place 2 ml of egg albumin in each of the two (2) test tubes.
2. Add 10 drops of concentrated nitric acid to the first test tube allowing the acid to
slide down the side of the tube.
3. In the second test tube, add 10 drops of concentrated sulfuric acid.
4. Note the color changes at the junction of the protein – acid layer.

Result:

Nitric acid –

Sulfuric acid –

Result:

3. By alkaloidal reagents
Reagents:
5% potassium ferrocyanide solution, acetic acid

Procedure:
1. Place 2 ml of the egg albumin solution in a test tube.
2. Acidify with three (3) drops of concentrated acetic acid.
3. Add five (5) drops of 5% potassium ferrocyanide solution.
4. Shake well.
5. Observe.

Result:
Color Reaction Tests

1. Biuret test
Reagent:
dilute CuSO4, dilute NaOH

Procedure:
1. Place 2 ml of the egg albumin solution in a test tube.
2. Add 10 drops of dilute NaOH and 10 drops of dilute CuSO4 solution.
3. Shake the mixture.
4. Observe.

Result:

2. Xanthroproteic test
Reagent:
concentrated nitric acid, NH4OH

Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add five (5) drops of concentrated nitric acid.
3. Heat in a water bath for 5 minutes.
4. Note the color change of the solution.
5. Cool the mixture and add 3 drops of concentrated NH4OH.
6. Observe the color change.

Result:

3. Million’s test
Reagent:
Million’s Reagent

Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add five (5) drops of Million’s reagent.
3. Heat in a water bath for five (5) minutes.
4. Observe.

Result:

4. Glyoxylic acid test (Hopkin’s – Cole Test)

Reagent:
glyoxylic acid reagent, concentrated sulfuric acid.
Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add 10 drops of glyoxylic acid reagent. Shake well.
3. Carefully pour along the side of the test tube 1 ml of concentrated sulfuric acid.
4. Observe for the formation of a layer under the lighter liquid.

Result:

5. Ninhydrin test
Reagent:
solid sodium acetate, 0.5% ninhydrin solution

Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add a pinch of solid sodium acetate to neutralize the solution.
3. Add 3 drops of 0.5% Ninhydrin solution then heat in a water bath for 3 minutes.
4. Cool the mixture.
5. Observe.

Result:

6. Sakaguchi test
Reagent:
20% NaOH, alphanapthal solution, bromine water

Procedure:
1. Place 2 ml of egg albumin solution in a test tube.
2. Add 10 drops of freshly prepared alpha-napthol solution.
3. Shake then add 10 drops of freshly prepared bromine water.
4. Observe.

Result:

7. Reduced sulfur test

Reagent:
20% NaOH, lead acetate solution

Procedure:
1. Place 2 ml of egg albumin a test tube.
2. Add 10 drops of 20% NaOH and 10 drops of lead acetate solution.
3. Boil in a water bath for 5 minutes.
4. Observe.

Result:

Question:

In the color reaction tests performed, indicate the positive result and the group
responsible for as indicated by this result on the table below.

Color Reaction Test Positive Result Group Responsible

Biurent test

Xanthoproteic test

Million’s Ttst

Glyoxylic acid test

Ninhydrin test

Sakaguchi test

Reduced sulfur test

5. What are peptides? Do all proteins possess peptide bonds?

6. Differentiate essential from non-essential amino acids. Give all examples under
each type of amino acid.
Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
Enzymes

An enzyme is a protein synthesized by a living cell that catalyzes or speeds up chemical

reaction. In the absence of enzymes therefore, any chemical reaction slows down.

Preparation of Enzyme Samples:

Weigh 5 grams of powdered starch and dissolve this in 50 ml of water. Heat 150 ml of
water to boiling and add the dissolved starch in it while stirring. Again, heat to boiling
and cool.

Collection of Saliva

Chew a small piece of paraffin to stimulate salivary secretion. Collect the saliva I in a
test tube with filter paper moistened with distilled water.

A. Action of salivary amylase

Reagents: dilute iodine solution, Benedict’s solution
 Procedure:
1. Place 5 ml of starch solution in a test tube.
2. Add five (5) drops of filtered saliva.
3. Shake the mixture.
4. Place a drop of the starch-saliva mixture on a spot place and add one (1) drop
of dilute iodine solution to it. Observe the change in color after 15
minutes.
5. Warm the remaining starch-saliva mixture in a water batch at 35 0C while
performing the next step.
6. Repeat the iodine test at 1 minute, 2 minute, 3 minute and 5 minute
intervals, noting the color change.
7. Transfer five (5) drops of the remaining saliva-starch mixture in a test tube.
8. Add 1 ml of Benedict’s solution.
9. Place in a water bath for 3 minutes. Record the result.

Result:

Subject the remaining mixture in the test tube to Benedict’ test.

Result:

Test for Dilution on Enzymes Activity

Reagents: iodine solution, Benedict’s solution

Procedure:
1. Place 5 ml of water in each of 6 test tubes and number the test tube from 1 to 6.
2. To test tube 1, add 1 ml of filtered saliva and shake the mixture. Transfer 1 ml
from test tube 1 to test tube 2, then 2 to 3, 3 to 4, 4 to 5, 5 to 6, making 6 different
solutions gradually with decreasing concentrations.
3. To each of the test tubes, add 1 ml of starch solution and shake well.
4. Place the test tube in a water bath at a temperature of 40 0C for 10 minutes then
divide the set samples into two equal portions.
5. Treat the first portion with 5 drops of Iodine solution and 1 ml Benedict’s
solution for the second portion.

Result:
Test Tube 1: Iodine solution

Benedict’s solution

Test Tube 2: Iodine solution

 Benedict’s solution

Test Tube 3: Iodine solution

Benedict’s solution

Test Tube 4: Iodine solution

Benedict’s solution

Test Tube 5: Iodine solution

Benedict’s solution

Test Tube 6: Iodine solution

Benedict’s solution

Effect of Temperature on Enzymes Activity

Reagent: iodine solution

Procedure:
1. Place 5 ml of starch solution in each of the 6 test tubes.
2. Label the test tubes 1 to 6.
3. Place test tube 1 in an ice bath at 5 0C, test tube 2 in an ice water at 10 0C, test tube
3 in a cold water bath at 20 0C, test tube 4 at 30 0C, test tube 5 in a water bath at 40 0C,
and test tube 6, at 600C. Add 5 drops of saliva to test tube 1, 2, 3, 4, and 6, and 5
drops of boiled saliva to test tube 5. Shake all test tubes well then add one drop of
iodine to each test tube. Observe.

Result:
Test Tube 1

Test Tube 2

Test Tube 3

Test Tube 4

Test Tube 5

Test Tube 6
B. Action of Catalase
Preparation of Potato Extract:
Weigh 25 g. of fresh potato, peel and slice it thinly. Pound it in a mortar and add
100 ml. of distilled water. Filter the solution. (Use the filtrate for the following tests.)

Effect of Concentration
Reagents: 3% H2O2, 1% H2O2
Procedure:
1. Place 1 ml. of potato extract to two (2) separate test tubes.
2. Place the two (2) test tubes in a water bath at 350C for 5 minutes.
3. Add 15 drops of 3% H2O2 on the 1st test tube and 15 drops of 1% H 2O2 on the
second test tube.
4. Note the time required for bubbles to appear.

Result:

Effect of Temperature
Reagent: 3% H2O2

Procedure:
1. Place 1 ml. of potato extract into two (2) separate test tubes.
2. Place the first test tube in an ice bath, and the second test tube to a water bath at
1000C.
3. Treat the two (2) test tubes with 10 drops of 3% H 2O2. Note the time required for
bubbles to appear.

Result:

Effects of pH
Reagent: 10% NaOH, 3% H2O2

Procedure:
1. Place 1 ml. of potato extract in a test tube. Determine the pH.
2. Then add five (5) drops of 10% NaOH. Determine the pH.
3. Warm the extract in a water bath at 350C. Add 10 drops of 3% H2O2.
4. Note the time required for bubbles to appear.

Result:
Questions:

1. What is an amylolytic enzyme?

2. What is the effect of dilution on enzyme activity?

3. What is meant by Q10?

4. What is the effect of temperature on enzyme activity?

Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
Digestion

Digestion is the process of changing food into simple forms. This process is
catalyzed by enzymes which are secreted by the glands found either within the
different regions of the digestive tube or outside the tube emptying their secretions into
the tube through their respective ducts.

Collection of Saliva

Refer to the experiment on enzymes.

Extraction of Gastric Enzyme:

Turn a pig’s stomach inside out, wash it with water, and strip off the mucous
membrane. Mince the membrane, place it in a clean bottle and completely submerge it
in glycerol. Stir frequently and allow to stand at room temperature for two (2) days.
Decant the glycerol portion into a small flask and set aside.

Extraction of Pancreatic Enzyme:

Remove the fats from a pig’s pancreas and grind them in a meat grinder. Place
the pancreatic tissue in a flask and add 100 ml. of water and 50 ml. of 95% alcohol.
Shake well and allow to stand for two (2) days. Strain the alcoholic extract through
cheesecloth. Test the pH of the extract, and neutralized it.

Preparation of Intestinal Extract:

Scrape the mucous membrane of the washed duodenum and jejunum of a pig’s
intestine. Grind the scrapings with washed sand in a mortar, transfer it to a flask, and
add 50 ml. of 1% NaCl and 5 ml. of toluene. Allow to stand for two (2) days at room
temperature. Shake the mixture frequently. Then strain the mixture with a cheesecloth
and store the extract in a refrigerator.
Digestion of Carbohydrates

1. By Salivary amylase
Reagents: iodine solution, Benedict’s solution.

Procedure:
1. Place 5 ml. of 1% starch solution in a test tube.
2. Warm in a water bath at 40 0C, maintaining the temperature throughout the
experiment.
3. Add 1 ml. of saliva to the mixture and mix thoroughly.
4. At one- minute interval, transfer five (5) drops to one depression on the spot
plate, and 5 drops of Benedict’s solution. Continue the test until the starch solution
no longer gives a color reaction with iodine.
5. Treat the remaining mixture in the test tube with 10 drops of Benedict’s solution.
6. Place the test tube in a water bath for three (3) minutes.
7. Observe.

Result:

2. By Pancreatic Amylase:
Reagents: iodine solution, Benedict’s solution

Procedure:
1. Place 5 ml. of starch solution in 2 ml. of pancreatic extract in a test tube.
2. Shake well and place in a water bath at 400C for 30 minutes.
3. Divide the mixture into two (2) equal portions.
4. To the first test tube add a drop of iodine solution and to the second test tube
add five (5) drops of Benedict’s solution.
5. Observe.

Result:

3. By Intestinal Disaccharidase:
Reagents: Benedict’s solution, 1% sucrose, 1% maltose, 1% lactose

Procedure:
1. Place 1 ml of 1% sucrose solution in each of the two test tubes.
2. To test tube 1, add 1 ml of intestinal extract.
3. To test tube 2, add 1 ml of previously boiled intestinal extract.
4. Place the two test tubes in a water bath maintained at 400C for 30 minutes.
5. Add five (5) of Benedict’s solution to the two test tubes. Observe.
6. Repeat the procedure using 1% lactose and 1% maltose.

Result:

Sucrose with intestinal extract

Sucrose with boiled intestinal extract

Lactose with intestinal extract

Lactose with boiled intestinal extract

Maltose with intestinal extract

Maltose with boiled intestinal extract

Digestion of Proteins:

1. By Gastric Enzyme:
Reagent: 0.4% HCI, 1% Na2CO3, 1% CuSO4, toluene

Procedure:
1. Place the following in four (4) separate test tubes.
A. 5 ml of gastric extract
B.5 ml of 0.4% HCI
C. 5 ml of gastric extract + 3 ml of 0.4% HCI
D. 5 ml of gastric extract + 3 ml of 1% Na2CO3

Add equal slices of coagulated egg white to each tube and place the test tubes in a water
bath at 400C for two (2) hours. Add four (4) drops of toluene to each test tube and store
until next laboratory period. Determine the extent of protein digestion by noting the
size of the coagulated egg white.

Observation:

A. Test tube 1

B. Test tube 2

C. Test tube 3
 D. Test tube 4
Place 1 ml. of the supernatant fluid from each test tube in separate tubes. Neutralize
test tube B and C with solid Na2CO3. In all the test tubes, add 1 ml. of 1% NaOH and
two (2) drops of 1% CuSO4.

Result:

Test tube A

Test tube B

Test tube C

Test tube D

2. By Pancreatic Enzyme:
Reagents: 0.4% HCI, 1% Na2CO3

Procedure:
1. Place the following in four (4) different test tubes.
A. 5 ml of gastric extract
B. 5 ml of 0.4% HCI
C. 5 ml of gastric extract + 3 ml of 0.4% HCI
D. 5 ml of gastric extract + 3 ml of 1% Na2CO3

Place equal sizes of coagulated egg white in each test tube and place them in a water
bath at 400C for one and a half hours. Add three (3) drops of toluene to all test tubes
and set aside until the next laboratory period. Then add 1 ml of CUSO 4 to all mixtures.

Result:

Test tube A

Test tube B

Test tube C

Test tube D
3. By Intestinal Enzymes:
Reagents: 1% Na2CO3, 0.4% HCI

Procedure:
1. Prepare four (4) test tubes as follows:
A. 5 ml of water
B. 5 ml of intestinal extract + 1 ml of 1% Na2CO3
C. 5 ml of boiled intestinal extract + 1ml of 1% Na2CO3
D. 5 ml of boiled intestinal extract + 1 ml of 0.4% HCI

Add 1 ml of milk to each test tube and mix thoroughly. Divide the contents of the four
(4) test tubes equally and set aside the remaining four test tubes as control tubes. Place
the test solutions in a water bath at 40 0C for one hour. Add 1 ml of CUSO 4 for both
incubated tubes and the control tubes.

Result:
Incubated tubes:
Test tube A

Test tube B

Test tube C

Test tube D

Control Tubes:

Test tube A

Test tube B

Test tube C

Test tube D

4. Intestinal Digestion by Proteases:

Reagent: 1% Na2CO3

Procedure:
1. Prepare four(4) test tube as follows:
A. 5 ml of 1% peptone
B. 5 ml of 1% peptone
 C. 5 ml of 1% albumin
D. 5 ml of 1% casein solution

2. Prepare another set of four (4) test tubes containing the above solution and make
these as the control tubes.
3. Add three (3) drops of 1% Na2CO3 to 10 ml of the intestinal extract.
4. Add two (2) ml of the alkaline extract to test tube A, C, and D. Boil the remaining
extract and add to test tube B.
5. Place all test tubes in a water bath at 400C for one and a half hours.
6. Add 1 ml of CUSO4 in all the mixtures.

Result:
Incubated tubes:
Test tube A

Test tube B

Test tube C

Test tube D

Control tubes:

Test tube A

Test tube B

Test tube C

Test tube D

Digestion of Lipids:
Reagents: 0.05N NaOH, phenolphthalein

Procedure:
1. Place the following in each of the four (4) test tubes
A. 1 ml coconut oil + 4 ml pancreatic extract + 5 ml of H2O
B. 1 ml coconut oil + 9 ml of water
C. 1 ml coconut oil + 2 ml bile + 7 ml of H2O
D. 1 ml coconut oil + 2 ml bile + 4 ml pancreatic extract + 3 ml of H2O
2. Shake the test tubes well and place in a water bath at 400C for one and a half hours.
3. Add a drop of phenolphthalein to each test tube.
4. Add drop by drop of 0.05 N NaOH using a syringe until the solution turns to a faint
pink color.
5. Record the amount of 0.05 N NaOH use for each test tube.

Result:

Test tube A

Test tube B

Test tube C

Test tube D

Questions:

1. Give the effect of the following:

A. Boiling the enzyme on its activity

B. Bile on fat digestion

C. HCI on gastric digestion

2. Proteolytic enzymes are secreted as proenzymes. What is the significance of this?

3. Discuss briefly the activation of:

A. Pepsinogen

B. Trypsinogen

C. Chymotrypsinogen

4. In these experiments on digestive enzymes, what factors influenced the rate of

enzyme-catalyzed reaction?

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
 Nutrition

The science of nutrition tries to find ways and means of different qualitative and
quantitative requirements in order to promote good health.

1. Devise a diet for weight reduction. Explain how each item in the diet will attain this
goal.

2. Why do various food proteins have different nutritional values?

3. What are the nutritional consequences of a low – fat diet?

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
 VITAMINS

Vitamins are organic nutrients which are required in small quantities for normal
metabolism and which they cannot be synthesized by the body in adequate amounts.
Vitamins maybe classified into water- soluble vitamins and fat- soluble vitamins. Fat
soluble vitamins include the vitamins A, D, E, and K, and the water soluble vitamins
include the vitamins of the B complex, and C.
Determination of the amount of vitamins present in foods, blood and urine is
necessary to evaluate the nutritional status or wellness of man. In this experiment, we
will have preparation and properties of vitamin A and vitamin C.

FAT SOLUBLE VITAMINS:

A. Preparation of Crude Carotene Extract:
Reagents: 95% ethanol, petroleum ether, carrots or other yellow sources

Procedure:
1. Cut 20 g. of previously washed carrots into thin pieces.
2. Macerate into a clean mortar and pestle.
3. Add 100 ml. of hot 95% ethanol solution.
4. Mix thoroughly, then transfer in a clean flask.
5. Set aside for half an hour. Decant the yellow solution.
6. Extract with 25 ml. of petroleum ether, and let it stand.
7. Separate the two layers with separatory funnel.
8. Concentrate the extracts in a vacuum just to dryness.
9. Dissolve the oily residue with 5 ml of petroleum ether and keep the extract in a
tightly stoppered container in the dark.

B. Chromatographic Separation of Carotenes:

Reagents: silica, magnesium oxide, anhydrous sodium sulfate,

Materials: chromatographic columns

Procedure:
1. Carefully packed a column of 1:1 ratio of silica: MgO a depth of 3 inches.
2. Add a few ml. of petroleum ether to cover the column with liquid careful not to
disturb the surface of the column.
3. Gently pour 1 cm. Layer of anhydrous sodium sulfate ontop of the column.
4. Immediately, pipette 3 ml. of the carotene solution on to the column.
5. Observe for the formation of a well developed bands of color separated by clear
white bands.
6. Separate the desired compound by allowing it to flow from the bottom of the
column with the use of 10% ethanol in petroleum ether as the solvent.
7. Use vials to contain the fractions being separated and label it properly.
WATER SOLUBLE VITAMINS:
A. Ascorbic Acid Saturation Test:
Reagents: ascorbic acid, toluene, 10% meta-phosphoric acid, 2,6 dichlorophenol-
indophenol.

Procedure for Urine Sample Collection:

1. One week before the test, the experimental student is given 100 mg. ascorbic acid
daily.
2. The day before the laboratory period, both control and experimental student are
given 500 mg. of ascorbic acid.
3. The 24- hour urine is collected using toluene as preservative.
4. The total volume is measured and 200 ml. of urine is brought to the laboratory the
following day for ascorbic acid determination.
Procedure for Ascorbic Acid Determination

1. Acidify 5 volumes of urine with 1 volume of 10% meta-phosphoric acid and place
this in a burette.
2. Pipette 1 ml. of 0.02% of 2,6 dichlorophenol-indophenol in an evaporating dish.
3. Titrate by constant and dropwise addition of the acidified urine. Blue color of the
dye changes to deep red upon the addition of urine.
4. Disappearance of thread color indicates end point of the reaction.
5. Calculate the amount of ascorbic acid present.

Wt. Ascorbic acid in mg. = V x S x D

where:
V = volume of the acidified urine
S = equivalent ascorbic acid/ml of dye
(1 ml. of dye = 0.066 mg. of ascorbic acid)
D = dilution factor

QUESTIONS:

1. Why is petroleum ether use as a solvent for extraction?

2. What is the rationale behind the in vacuum drying of the petroleum ether extract?

3. What are some precautionary measures in the preparation of chromatographic

columns?

4. What is the purpose of adding anhydrous sodium sulfate in the experiments?

5. Carotene structure can be divided into eight repeating 5-carbon units. What is the
name and structure of this unit and to what class of compounds do carotene belong?
Name: Date Performed:
Course/Year/Section: Rating:

Experiment No.
Blood

The blood, the circulating medium of the body is composed of a liquid portion, the
plasma, formed elements, the red blood cells or erythrocytes, the white blood cells or
leucocytes, and the blood platelets or thrombocytes. The blood reflects the overall
metabolism of the tissue, thus, qualitative and quantitative analyses of its components
are very useful for the diagnosis of many pathological conditions.

Test for Blood

1. Guaiac Test
Reagents: 2% Guaiac alcoholic solution, H2O2

Procedure:
1. Add a new drop of 2% guaiac solution to 1 ml of blood until turbidity sets in.
2. Then add H2O2 drop by drop

Result:

2. Benzidine Test
Reagents: 3% H2O2, 1% Sodium acetate

Procedure:
1. To 3 ml of freshly prepared benzidine, add a few drops of diluted blood.
2. Then add 1 ml of 3% H2O2 and 1% sodium acetate.

Result:

3. Hemin Test
Reagents: NaCl crystals, Glacial Acetic acid

Procedure:
1. Place a small drop of blood on a glass slide and allow it to dry.
2. Add a small crystal of NaCl and a drop of glacial acetic acid.
3. Cover with a cover slip and heat to boiling over a low flame.
4. Examine under the microscope.

Result:

Determination of Various Constituents

Preparation of defibrinated blood

Place a freshly drawn blood into a clean test tube and stir vigorously with a glass rod
until a light straw- colored liquid separates upon standing. Decant.

Preparation of Test Solution

Procedure
1. Place 40 ml of water in a breaker and add 5 drops of 10% acetic acid.
2. Boil. Add 10 ml of defibrinated blood, stirring while adding to the mixture.
3. Continue boiling until a coagulum forms and the supernatant liquid becomes clear.
Filter.
4. Evaporate the filtrate ½ its original volume.
5. Divide the mixture into 3 parts for the following test:

1. Benedict’s Test
Reagents: 10% Na2CO3, Benedict’s solution

Procedure:
1. Neutralized 1 ml of the test solution with 10% Na2CO3 solution.
2. Add 5 ml of Benedict’s solution and boil in a water bath. Observe.

Result:

2. Test for Chlorides

Reagents: Concentrated Nitric Acid, AgNO3

Procedure:
1. To 2 ml of the test solution, add 3 drops concentrated nitric acid and 1 ml of AgNO 3
solution. Observe.

Result:

3. Test for Phosphates

Reagents: Concentrated HNO3, Ammonium Molybdate
Procedure:
1. Mix 5 ml of the test solution with 3 drops of concentrated HNO3.
2. Gently boil the mixture and add dropwise 2 ml of ammonium molybdate. Observe

Result:

Blood Coagulation

Procedure: Prepare a series of small test tube as follows:

Tube 1 – to contain a small amount of powdered Potassium Oxalate (20 to 50
mg.)
Tube 2 – to contain powdered Sodium Citrate
Tube 3 – to contain Sodium Flouride (small amount of powdered salt)
Tube 4 – to contain a few milligrams of heparin
Tube 5 – Empty
Tube 6 – Cooled ice
Tube 7 – paraffin or oil

Distribute without delay approximately 2 ml of blood into each tube. After mixing the
contents of the tube 1, 2, 3 and 4 gently thoroughly to dissolve the powder, set all tubes
on a rack. Note the time required for the blood to clot.

Result:

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

Tube 6

Tube 7

Note: If the blood does not clot, Explain the cause of this reaction.
Blood Typing

The determination of the blood type of an individual is dependent on the

antigen/agglutinogen present in his RBC. In the ABO series of blood type, the method
used in the determination of blood type of an individual is by means of the typing sera.
Procedure: Clean the ball of one finger with alcohol and puncture the skin by means of
a blood lancet. Place a drop of blood, one on both sides of a glass slide. Add one drop
of anti – A serum to one and anti – B serum to the other. Mix the serum and the blood
and observe for agglutination.

Result:

Anti – A serum + blood =

Anti – B serum + blood =
Possible agglutinogen (s) present =
Blood type is =

Questions:

1. Differentiate blood plasma from serum.

2. Is guaiac or benzidine test reliable to test the presence of blood? Explain

3. Give several examples of blood anti – coagulants and give their mechanisms of
action?

4. Aside from the A and B antigens, what other blood antigens are present.

Name: Date Performed:

Course/Year/Section: Rating:

Experiment No.
Urine

The urine is an aqueous solution of inorganic and organic substances, which are either
waste product of metabolic processes in the body, or derived from the food taken in.
The composition of the urine is important in determining the physiological and
pathological conditions of an individual.
Collection of Urine Samples

Samples of urine collected at different intervals during the day usually show
considerable variation in composition; hence the analysis of specimen collected at
random has limited significance or none at all. As a rule, the quantitative analysis of
urine is performed on a mixed 24 – hour sample. This may be collected as follows: the
subject empties his bladder at a fixed time in the morning, discarding the urine. From
this time on, all urine, including that voided at exactly the same hour the following
morning is collected in clean bottle containing 10 to 20 ml of toluene to prevent bacterial
solution.

Analysis of Normal Constituents

Inorganic Contituents:

1. Calcium
Reagent: 2% potassium oxalate solution

Procedure:
1. Place 2 ml of urine sample in a test tube.
2. Add three (3) drops of 2% potassium oxalate solution.
3. Observe.

Result:

2. Magnesium
Reagent: 10% Ammonia water

Procedure:
1. Filter the solution on calcium determination on the above procedure.
2. To the filtrate add five (5) drops of ammonia water.
3. Set aside.
4. Observed.

Result:

3. Chlorides
Reagents: HNO3 solution, AgNO3 solution

Procedure:
1. Place two (2) ml of urine sample in a test tube.
2. Add three (3) drops of HNO3.
3. Then add five drops of AgNO3 solution.
4. Observe.

Result:
4. Sulfates
Reagent: HCI, 10% BaCl2

Procedure:
1. Place two (2) ml of the urine sample in a test tube.
2. Acidify with five (5) drops of HCl.
3. Then add ten (10) drops of 10% BaCl2 solution.
4. Observe.

Result:

Organic constituents

1. Urea
Reagents: 20% NaOH, 0.5% CuSO4

Procedure:
1. Evaporate ten (10) ml of the urine sample until its volume is reduced to three (3) ml.
2. Cool.
3. Add two (2) ml of 20% ml NaOH. Shake.
4. Add five (5) drops of dilute CuSO4.
5. Observe.

Result:

2. Uric Acid
Reagents: 25% HCI, concentrated HNO3, NH4OH

Procedure:
Isolation: Place 50 ml of filtrated urine in a beaker and add eight (8) drops of
concentrated HCI. Set aside until the next laboratory period. Observed that the uric
acid crystals settle. Filter and test the crystals.

Murexide Test:
1. Place a small amount of uric acid crystals in an evaporating dish.
2. Add two (2) drops of concentrated HNO3.
3. Evaporate to dryness in a water bath.
4. A reddish or yellowish residue will remain on the dish.
5. Cool the residue and add one (1) drop of dilute NH4OH.
6. Observe.

Result:

Draw uric acid crystals as seen under the microscope.

3. Creatinine

A. Jaffe’s Reaction
Reagents: saturated picric acid solution, 10% NaOH

Procedure:
1. Place three (3) ml of urine in a test tube.
2. Add one (1) ml of saturated picric acid solution. Shake.
3. Add five (5) drops of 10% NaOH to make the solution alkaline.
4. Observe.

Result:

a. Nitroprusside Test
Reagents: sodium nitroprusside solution, 10% NaOH.

Procedure:
1. Place two (2) ml of urine in a test tube.
2. Add five (5) drops of freshly prepared sodium nitropusside solution.
3. Add 5 drops of 10% NaOH to make it alkaline.
4. Observe.

Result:

B. Indican
A. Obermayer’s Test
Reagents: obermayer’s reagents, CCL4

Procedure:
1. Place three (3) ml of urine in a test tube.
2. Add two (2) ml of Obermayer’s reagent.
3. Then add one (1) ml of CHCl3 and shake the mixture.
4. Observe.
Result:

a. Jaffe’s Test
Reagents: concentrated HCI, chloroform, calcium hypochlorite solution

Procedure:
1. Place five (5) ml of urine in a test tube.
2. Add ten (10) drops of concentrated HCI.
3. Add three (3) ml of chloroform and five (5) drops of calcium hypochlorite solution.
4. Shake the mixture thoroughly.
5. Observe.

Result:
Analysis of Abnormal Constituents

1. Protein (Albumin)
A. Coagulation Test
Reagent: 2% Acetic Acid

Procedure:
1. Heat two (2) ml of urine in a test tube at an angle of 450.
2. Boil the upper half by passing it over the flame. If turbidity develops, it may be due
to protein or phosphate.
3. Acidify with two (2) drops of 2% acetic acid. If turbidity disappears, it is due to
phosphate. If protein is present, turbidity will not disappear, but will become more
flocculent.

Result:

B. Heller’s Ring Test

Reagent: concentrated HNO3

Procedure:
1. Place 2 ml of urine in a test tube.
2. Add 2 ml of concentrated HNO3 by means of a pipette, add along the side of the test
tube, taking care not to mix contents.
3. Formation of a white zone (white ring) or precipitated protein at the junction of the
two liquids indicate the present of protein.

Result:
a. Robert’s Test
Reagent: Robert’s reagent

Procedure:
1. Place 2 ml of urine in a test tube.
2. Add by means of a pipette 1 ml of Roberts reagent allowing it to occupy the zone
beneath the urine sample.
3. Formation of a white ring at the zone of contacts, indicates that albumin is present.

Result:

B. Glucose
Benedict’s Test
Reagent: Benedict’s solution

Procedure:
1. Place 2 ml of Benedict solution in a test tube.
2. Boil the solution in a water bath for 3 minutes.
3. Add 1 ml of urine and mix thoroughly.
4. Cool. The amount of precipitate and its color (green or red) will depend on the
quantity of glucose present in the urine.

Result:

C. Acetone Bodies
A. Legal’s Test
Reagents: 5% sodium nitroprusside, NaOH

Procedure:
1. Place 2 ml of urine sample in a test tube.
2. Add 5 drops of freshly prepared sodium nitroprusside solution.
3. Mix thoroughly and add three (3) drops of dilute NaOH.
4. A ruby red color is produced if acetone is present.
5. Acidify with three (3) drops of dilute acetic acid.
6. If acetone is present, the red color is intensified, if absent, it becomes yellow.

Result:

B. Bile and Bile Pigments

Gmelin’s Test
Reagent: concentrated HNO3

Procedure:
1. Place 4 ml of concentrated HNO3 in a test tube.
2. By means of a pipette, deliver down the side of the test tube the 3 ml of urine.
Avoid mixing.
3. Colored rings will be formed (green nearest the urine, blue, violet, red and reddish
yellow nearest the acid) if bile is present.

Result:

C. Bile Acids
Pettenkofer’s Test
Reagents: 5% sucrose solution, concentrated H2SO4

Procedure:
1. Place 3 ml of urine in a test tube.
2. Add five (5) drops of 5% sucrose solution.
3. Add 3 ml of concentrated H 2SO4 into the test tube by pouring at the side of the test
tube in an inclined position.
4. A red ring is observed at the point of contact to indicate the presence of bile acids.

Result:

D. Blood
Benzidine Reaction
Reagents: benzidine solution in glacial acetic acid, H2O2

Procedure:
1. Place 3 ml of urine in a test tube.
2. Boil in a water bath and cool.
3. Add 2 ml. of saturated benzidine solution.
4. Add 1 ml of 3% H2O2
5. A blue or green color is produced after about 10 minutes if blood is present.

Result:

Sedimentary Constituents
Procedure:
1. Centrifuge about 10 ml of urine for 30 minutes.
2. Discard the supernatant fluid or the centrifugate.
3. Place a drop of the sediments on a dry slide.
4. Examine them under the LPO and NPO.
5. Identify and draw the sediments seen.

Urinary sediments may be divided into:

1. Organized sediments like:
A. Calcium oxalate crystals – colorless
B. Urate crystals – amorphous materials
2. Unorganized sediments like
A. Epithelial cells – flat cells with prominent nucleus
B. Red Blood cells – circular cells with clear center
C. Pus cell – dead blood cell usually bigger than red blood cell,
granular and are nucleated.
D. Microorganisms –
E. Cast – hardened mucus, cylindrical transparent bodies.

Questions:

1. What pathological conditions are indicated if the following substances are

present in urine?
A. Glucose –

B. Acetone bodies –

C. Albumin –

D. Bile pigments –

E. Blood

2. Why should benzidine reagent be freshly prepared when used in some tests?

3. What is the most important constituent of the urine?

4. What hormone is associated with the amount of urine produced? Give the
mechanism of its action.
ISOLATION AND CHARACTERIZATION OF PROTEIN COMPONENT FROM THE
FOLLOWING:
A. Isolation of Casein
1. Weigh 50 g of skimmed milk.
2. Heat in a water bath with constant stirring until the temperature reaches 400.
3. Add 12 drops of glacial acetic acid with constant stirring.
4. Observe the formation of precipitate.
5. Filter the solution using cheesecloth.
6. Squeeze the cheesecloth to remove excess water.
7. Transfer the precipitate from the cheesecloth to an Erlenmeyer flask.
8. Add 25 ml of 95%ethanol to the precipitate and stir for 5 minutes.
9. Allow the precipitate to settle. Decant.
10. To the precipitate wash with 10 ml of diethyl ether and 10ml of ethanol.
11. Discard the washing, collect and dry the precipitate.