For the pre-task of this module, you need to perform and decant the cell suspension onto the

a home-based experiment (to be done individually) cheesecloth. Let the solution filter into the
designed to isolate the DNA from a fruit source. For glass container, while gently squeezing the
this experiment, you will be utilizing materials cloth to obtain the maximum yield, leaving
available at your home, and your resourcefulness as the cellular debris behind. Cool the solution
a pharmacy student. to room temperature.
5. To the viscous DNA-containing aqueous
As a reminder, always practice safety when solution, slowly add 5 tablespoons of cold
performing experiments even at home. Wear your 70% ethanol (pre-chill the alcohol to be
personal protective equipment (PPE) when used), taking care that when you add the
performing this experiment. alcohol, it flows along the side of the glass
MATERIALS, APPARATUS, AND REAGENTS container, settling on top of the aqueous
Test Sample solution and forming a layer.
Banana or onion 6. The next step is critical. Insert a wooden
Materials & Apparatus stick (e.g. chopsticks) gently into the
Clean transparent glass jar/bottle solution reaching just below the interface.
Funnel Rotate (do not stir) the glass rod in one
Hot water bath direction (clockwise or counterclockwise).
Ice-water bath The rotation spools all the DNA precipitate
Tablespoon onto the glass rod.
Teaspoon 7. Air dry the spooled DNA on the wooden
Wooden stick (e.g. chopsticks) stick. Describe its appearance, color, odor
Cheesecloth or any clean filter cloth and texture.
Mortar & pestle (any material for pounding and 8. Make/Draw a summary and draw
grinding) conclusion.
Reagent
Distilled/mineral water CHARACTERIZATION OF DNA AND RNA
Liquid detergent or liquid dishwashing soap Image result for dna and rna
Salt
70% ethyl alcohol, pre-chilled

PROCEDURES
Extraction of DNA from fruit source (Banana, Kiwi,
Onion or Strawberry)

1. Cool a mortar in an ice bath. Place on it 1

medium-sized fruit of choice, diced. Grind
the fruit vigorously with a pestle for 5
minutes to disrupt the cells. A fruit mush
must form.
2. Using a hot water bath, warm 10-12
tablespoon of distilled water placed in a Nucleic acids are macromolecules that contain the
glass container to approximately 60°C. Add genetic information which can be passed on from
one and a half tablespoon of sodium one generation to another. These bioinformational
chloride and 1 tablespoon of liquid materials determine traits and make protein
detergent. Very slowly stir to dissolve but synthesis possible.
make sure that there are no bubbles
formed. After doing the pre-task, you have successfully
3. From the prepared saline-detergent isolated the DNA from the fruit source. After the
solution, get 3 tablespoons and transfer it isolation process, it must undergo characterization
to another glass container. Add the through simple qualitative chemical tests. Below are
previously prepared ground fruit mush. those tests used to identify and characterize the
Using a teaspoon, slowly stir the solution isolated DNA from the fruit source.
for 5 minutes, all the while maintaining the
temperature at approximately 60°C. Meanwhile, below also is the isolation procedure for
4. Prepare a filtration set-up. Add a RNA from the yeast source, followed by the
cheesecloth or clean filter cloth to a funnel qualitative chemical tests for the identification and
characterization of the isolated RNA.
 3. Add a pinch of potassium chlorate to the
PROCEDURES sample. Stir the mixture again using a glass
Acid Hydrolysis of DNA rod.
1. To the resuspended crude DNA, add 2.0 mL 4. Gently dry the solution over a Bunsen
of 1 M hydrochloric acid. flame.
2. Heat at 65°C for 60 minutes using a hot 5. Allow the sample to cool at room
water bath. temperature then moisten the dried
3. Allow the solution to cool. The hydrolyzed mixture with ammonium hydroxide
DNA will be used for chemical solution.
characterization. 6. Note the change in color of the solution.
Record result. Positive result is pink to
purple color.
Chemical Characterization of DNA Wheeler-Johnson test (Test for pyrimidine)
Dische Diphenylamine test (Test for deoxyribose) 1. Prepare a clean dry test tube. Place 20
1. Prepare a clean dry test tube. Place 20 drops of the hydrolyzed DNA. Label
drops of the hydrolyzed DNA. Label properly.
properly. 2. Add an excess bromine water dropwise
2. Add 40 drops of diphenylamine reagent. until the solution turns yellow.
Carefully shake the test tube. (Do not touch 3. Place the test tube in a boiling water bath
the bottom part of the test tube. It is hot.) until the solution turns light yellow or
3. Place the test tube in a boiling water bath colorless. Remove from heat and set aside
for 5 minutes. to cool.
4. Note the change in color of the solution. 4. Add an excess barium hydroxide solution
Record result. Positive result is blue color of dropwise while shaking until the red litmus
athe solution. paper turns to blue.
5. Note the change in color of the solution.
Ammonium molybdate test (Test for phosphate) Record result. Positive result is purple color
1. Prepare a clean dry test tube. Place 20 of the solution.
drops of the hydrolyzed DNA. Label
properly. Isolation of RNA from Yeast
2. Add 2.0 mL of 6 M nitric acid to the test 1. Weigh 5 grams of active dry yeast and place
tube. Carefully shake the test tube. it in a beaker.
3. Place the test tube in a boiling water bath 2. Add 5.0 mL of 1% sodium hydroxide and 25
for 5 minutes. Remove from heat and set mL of distilled water.
aside to cool. 3. Heat the mixture at 60°C for 15 minutes.
4. Neutralize the solution by adding 2.0 mL 6 Stir occasionally.
M sodium hydroxide. If precipitates appear, 4. Strain the heated mixture using a
filter the solution using a cheesecloth. cheesecloth.
5. Add 2.0 mL ammonium molybdate. Shake 5. Acidify the filtered mixture with glacial
the test tube. acetic acid until the blue litmus paper turns
6. Place the test tube in a boiling water bath to red.
for 5 minutes. Remove from heat and set 6. Heat the acidified mixture in a boiling water
aside to cool. bath until it is reduced to approximately 10
7. Add 5 drops of ascorbic acid solution to mL. Remove from heat and set aside to
solution. Shake the test tube. cool.
8. Let it stand for 10 minutes. Note the change 7. In a separate container, mix 20 mL of 95%
in color of the solution. Record result. ethanol and 0.2 mL of concentrated
Positive result is blue to purple color of the hydrochloric acid. Pour the solution into the
solution. reduced mixture and stir vigorously.
Murexide test (Test for purine) 8. Centrifuge the solution for 5 minutes and
decant the residue.
1. Prepare a clean dry evaporating dish. Place 9. Wash the residue twice with 2.0 mL of 95%
10 drops of the hydrolyzed DNA. ethanol and petroleum ether (1:1).
2. Add 5 drops of concentrated hydrochloric 10. Describe the isolated RNA.
acid. Stir using a glass rod.
Base Hydrolysis of RNA
 1. Place 2.0 mL of 0.3 N sodium hydroxide and 5. Allow the sample to cool at room
the isolated RNA in a test tube. temperature then moisten the dried
2. Heat for 60 minutes using a boiling water mixture with ammonium hydroxide
bath. solution.
3. Allow the solution to cool. The hydrolyzed 6. Note the change in color of the solution.
RNA will be used for chemical Record result. Positive result is pink to
characterization. purple color.
Wheeler-Johnson test (Test for pyrimidine)
Chemical Characterization of RNA 1. Prepare a clean dry test tube. Place 20
Bial’s test (Test for ribose) drops of the hydrolyzed RNA. Label
1. Prepare a clean dry test tube. Place 20 properly.
drops of the hydrolyzed RNA. Label 2. Add an excess bromine water dropwise
properly. until the solution turns yellow.
2. Add 2.0 mL of Bial’s reagent to the test 3. Place the test tube in a boiling water bath
tube. until the solution turns light yellow or
3. Place the test tube in a boiling water bath colorless. Remove from heat and set aside
for 5 minutes. Remove from heat and set to cool.
aside to cool. 4. Add an excess barium hydroxide solution
4. Note the change in color of the solution. dropwise while shaking until the red litmus
Record result. Positive result is green color paper turns to blue.
of the solution. 5. Note the change in color of the solution.
Record result. Positive result is purple color
Ammonium molybdate test (Test for phosphate) of the solution.
1. Prepare a clean dry test tube. Place 20
drops of the hydrolyzed RNA. Label INTRODUCTION TO NUCLEIC ACIDS
properly. INTRODUCTION TO NUCLEIC ACIDS
2. Add 2.0 mL of 6 M nitric acid to the test Nucleic acids are macromolecules that contains the
tube. Carefully shake the test tube. genetic information that that can be passed on from
3. Place the test tube in a boiling water bath one generation to another generation. These
for 5 minutes. Remove from heat and set bioinformational material determines traits and
aside to cool. makes protein synthesis possible. Nucleic acids can
4. Neutralize the solution by adding 2.0 mL 6 be found in the cytoplasm and nucleus of the cell.
M sodium hydroxide. If precipitates appear,
filter the solution using a cheesecloth.
5. Add 2.0 mL ammonium molybdate. Shake
the test tube.
6. Place the test tube in a boiling water bath
for 5 minutes. Remove from heat and set
aside to cool.
7. Add 5 drops of ascorbic acid solution to
solution. Shake the test tube.
8. Let it stand for 10 minutes. Note the change
in color of the solution. Record result.
Positive result is blue to purple color of the
solution.

Murexide test (Test for purine)

1. Prepare a clean dry evaporating dish. Place
10 drops of the hydrolyzed RNA. Nucleotides
2. Add 5 drops of concentrated hydrochloric
acid. Stir using a glass rod. DNA and RNA are polymers (in the case of DNA,
3. Add a pinch of potassium chlorate to the often very long polymers), and are made up of
sample. Stir the mixture again using a glass monomers known as nucleotides. When these
rod. monomers combine, the resulting chain is called a
4. Gently dry the solution over a Bunsen polynucleotide. Each nucleotide is made up of three
flame. parts: a nitrogen-containing ring structure called a
nitrogenous base, a five-carbon sugar, and at least
one phosphate group.
Figure 5.1. The basic structure of a nucleic acid.
Nitrogenous base
 organic molecules made up of nitrogen-
containing ring structures
 each nucleotide in DNA contains one of four
possible nitrogenous bases:
Figure 5.2. A DNA polynucleotide chain consists of a
string of nucleotides linked by phosphodiester
PURINE BASES PYRIMIDINE BASES
bonds.
Adenine (A) Thymine (T)
Guanine (G) Cytosine (C)
DEOXYRIBONUCLEIC ACID (DNA)
Uracil (U)
DNA is the chemical basis of heredity and is
organized into genes, the fundamental units of
Sugars
genetic information. It is involved in replication
 the five-carbon sugar in DNA is called
during cell division and gene expression by
deoxyribose, while in RNA, the sugar is
transcription. DNA commonly exists as a double
ribose
stranded molecule with a twisted double helix
 these two are very similar in structure, with
shape. The sugars and phosphates lie on the outside
just one difference: the second carbon of
of the helix, forming the backbone of the DNA
ribose bears a hydroxyl group, while the
(sugar-phosphate backbone). The nitrogenous bases
equivalent carbon of deoxyribose has a
extend into the interior in pairs; the bases of a pair
hydrogen instead
are bound to each other by hydrogen bonds. The
 in a nucleotide, the sugar occupies a central
two strands of the helix run in opposite directions,
position, with the base attached to its 1'C
meaning that the 5′ end of one strand is paired up
and the phosphate group (or groups)
with the 3′ end of its matching strand (antiparallel
attached to its 5'C
orientation). DNA base pairing is highly specific: A
can only pair with T, and G can only pair with C. The
Phosphate group
double helical structure may be separated when H-
 nucleotides may have a single phosphate
bonds is disrupted. Alteration of pH may lead to
group, or a chain of up to three phosphate
ionization of the nucleotide ionizes leading to DNA
groups, attached to the 5’C of the sugar
denaturation. Another factor that can cause
 in a cell, a nucleotide about to be added to separation of DNA double helix is temperature.
the end of a polynucleotide chain will bear a
series of three phosphate groups
 when the nucleotide joins the growing DNA
or RNA chain, it loses two phosphate groups
 therefore, in a chain of DNA or RNA, each
nucleotide has just one phosphate group

Polynucleotide chain
 A polynucleotide chain has directionality–
that is, it has two ends that are different
from each other. At the 5' end, or
beginning, the 5' phosphate group of the
first nucleotide in the chain sticks out. At
the 3' end, the 3' hydroxyl of the last
nucleotide added to the chain is exposed.
DNA sequences are usually written in the 5'
to 3' direction. As new nucleotides are
added to a strand of DNA or RNA, the
strand grows at its 3' end, with the 5'
phosphate of an incoming nucleotide
attaching to the hydroxyl group at the 3'
Figure 5.3. The double-helix structure of DNA.
end of the chain. This makes a chain with
each sugar joined to its neighbors by
For instance, if you know that the sequence of one
phosphodiester linkage.
strand is 5’-AATTGGCC-3’, the complementary strand
must have the sequence 3’-TTAACCGG-5’. This Figure 5.5. The mRNA, tRNA, and rRNA, involved in
allows each base to match up with its partner: protein synthesis.
Ribosomal RNA
 a major component of ribosomes, where it
helps mRNA bind in the right spot so its
sequence information can be read out
 some rRNAs also act as enzymes, in this
case, the formation of bonds that link
These two strands are complementary, with each amino acids to form a protein
base in one sticking to its partner on the other. The  RNAs that act as enzymes are known as
A-T pairs are connected by two hydrogen bonds, ribozymes
while the G-C pairs are connected by three hydrogen
bonds. Transfer RNA
 are also involved in protein synthesis, but
RIBONUCLEIC ACID (RNA) their job is to bring amino acids to the
RNA is essential for the synthesis of proteins. ribosome, ensuring that the amino acid
Information contained within the genetic code is added to the chain is the one specified by
passed from DNA to RNA to the resulting proteins. the mRNA
RNA is a single stranded nucleic acid composed of a  it consist of a single strand of RNA, but this
phosphate-ribose sugar backbone and the strand has complementary segments that
nitrogenous bases adenine, guanine, cytosine and stick together to make double-stranded
uracil (U). When DNA is transcribed into an RNA regions
transcript during DNA transcription, guanine pairs  this base-pairing creates a complex 3D
with cytosine (G-C) and adenine pairs with uracil (A- structure important to the function of the
U). molecule

THE CENTRAL DOGMA

DNA REPLICATION
DNA replication refers to the copying of a cell's DNA.
The process is said to be semiconservative, meaning
that each strand in the DNA double helix acts as a
template for the synthesis of a new, complementary
Figure 5.4. The single-stranded RNA translated from
strand. This process takes us from one starting
DNA.
molecule to two "daughter" molecules, with each
newly formed double helix containing one new and
Messenger RNA
one old strand. Cells need to copy their DNA very
 an intermediate between a protein-coding
quickly, and with very few errors (or risk problem
gene and its protein product.
such as cancer); to do so, they use a variety of
 if a cell needs to make protein, the RNA-
enzymes and proteins, which work together to make
polymerizing enzyme will make an RNA
sure DNA replication is performed smoothly and
copy, or transcript, of the gene’s DNA
accurately.
sequence
 the transcript carries the same information
THE CENTRAL DOGMA
as the DNA sequence of its gene however,
The direction of flow of information from DNA to
the base T is replaced with U
RNA to protein is termed as the central dogma. It
involves two major steps:
 transcription – the DNA sequence of a gene
is copied to make an RNA molecule, and
 translation – the sequence of the mRNA is
decoded to specify the amino acid sequence
of a polypeptide.
 three other “stop” codons signal the end of a
polypeptide
these relationships between codons and amino acids
are called the genetic code

Figure 5.9. The process of translation involves

Figure 5.6. The central dogma. translating the RNA to protein.
Transcription
one strand of the DNA that makes up a gene, called ISOLATION PROCEDURES FOR DNA AND RNA
the non-coding strand, acts as a template for the THE ISOLATION PROCEDURE
synthesis of a complementary RNA strand by the Below are the procedures in isolating the DNA from
enzyme RNA polymerase (this RNA strand is the onion and the RNA from yeast.
primary transcript)

Figure 5.10. Summary of the isolation procedure.

Figure 5.7. The process of transcription involves DNA EXTRACTION

"transcribing" the DNA template strand into a single- Step 1. Breaking cells open to release the DNA
stranded mRNA.
The cells in a sample are separated from each other,
Translation often by a physical means such as grinding or
the process of using information in an mRNA to build vortexing and put into a solution containing salt. The
a polypeptide is called translation positively charged sodium ions in the salt help
during translation, the nucleotide sequence of an protect the negatively charged phosphate groups
mRNA is translated into the amino acid sequence of that run along the backbone of the DNA.
a polypeptide
specifically, the nucleotides of the mRNA are read in A detergent is then added. The detergent breaks
triplets (groups of three) called codons down the lipids in the cell membrane and nuclei.
there are 61 codons that specify amino acids (one DNA is released as these membranes are disrupted.
codon is a "start" codon that indicates where to start
translation – Met) Step 2. Separating DNA from proteins and other
cellular debris

Figure To get a clean sample of DNA, it is necessary to

5.8. The remove as much of the cellular debris as possible.
codons This can be done by a variety of methods. Often a
denote protease (protein enzyme) is added to degrade DNA-
for a associated proteins and other cellular proteins.
specific Alternatively, some of the cellular debris can be
amino removed by filtering the sample.
acid.
Step 3. Precipitating the DNA with an alcohol
Finally, ice-cold alcohol (either ethanol or
isopropanol) is carefully added to the DNA sample.
DNA is soluble in water but insoluble in the presence
of salt and alcohol. By gently stirring the alcohol
layer with a sterile pipette, a precipitate becomes
visible and can be spooled out. If there is lots of Figure 5.12. Stepwise procedure in isolating the RNA
DNA, you may see a stringy, white precipitate. from yeast.

Step 4. Cleaning the DNA CHEMICAL TESTS AND ANALYSIS FOR DNA & RNA
CHEMICAL TESTS
The DNA sample can now be further purified Dische Diphenylamine Test
(cleaned). It is then resuspended in a slightly alkaline
buffer and ready to use. The Dische diphenylamine test detects the presence
of deoxyribose. The deoxyribose is converted to its
Step 5. Confirming the presence and quality of the derivative that binds to diphenylamine forming a
DNA blue-colored complex. The intensity of the blue color
is proportional to the concentration of the DNA
For further lab work, it is important to know the isolated from the sample.
concentration and quality of the DNA.

Optical density readings taken by a

spectrophotometer can be used to determine the Figure 5.6. Positive
concentration and purity of DNA in a sample. result for the
Alternatively, gel electrophoresis can be used to Dische
show the presence of DNA in your sample and give diphenylamine
an indication of its quality. test.

What can this DNA be used for?

Once extracted, DNA can be used for molecular
analyses including PCR, electrophoresis, sequencing,
fingerprinting and cloning.

Bial's Test
The Bial’s test detects the presence of pentoses,
such as ribose. When ribose is boiled in the presence
of hydrochloric acid, it yields aldehydes of furfural
type, that condenses to form green-colored
compound in the presence of orcinol.
Figure 5.11. Stepwise procedure in isolating the DNA
from onion.

RNA ISOLATION Figure 5.7. Positive result for

Yeast, Saccharomyces cerevisiae, is a unicellular the Bial's test.
fungus that contains 4% RNA by weight; it has low d-
ribonuclease and ribonuclease activity and can be
readily obtained in essentially pure form from this
source. Many proteins in human biology were first
discovered by their homologs in yeast. S. cerevisiae
was the first eukaryotic genome to be completely
sequenced. It was estimated that yeast shares 23%
of its genome with humans.
Ammonium molybdate Test
The ammonium molybdate test detects the presence
of phosphates. When the orthophosphate ion,
acidified with nitric acid, reacts with ammonium
molybdate, yellow-colored phosphomolybdic acid is
produced. Upon reaction with ascorbic acid,
phosphomolybdic acid is reduced forming intensely
blue to purple-colored complex.

Carbohydrates are macromolecules defined as

polyhydroxyketones or polyhydroxyaldehydes. They
can be determined by a set of qualitative chemical
tests, characterized by specific positive results, which
Murexide Test indicate the presence of such carbohydrate.
Murexide test detects the presence of purine. These
purine-containing compounds react with potassium INTRODUCTION TO CARBOHYDRATES
chlorate in the presence of hydrochloric acid to form
murexide or ammonium purpurate upon exposure to A carbohydrate is defined as an organic compound
ammonia. The product ammonium purpurate having a general formula CnH2nOn, that is, consists
appears as pink to purple in color. only of carbon, hydrogen and oxygen, where n
pertains to the number of carbon atom and the
hydrogen and oxygen have the same atom ratio as
that of water (H2O). Carbohydrates in general have
Figure 5.9. Positive either an aldehyde group (polyhydroxyaldehyde) as
result for the in glucose or a ketone group (polyhydroxyketone) as
murexide test. in fructose. Those containing an aldehyde group are
called as aldoses and those containing keto groups
are called ketose, as shown in Figure 6.2.
Carbohydrates are the most significant source of
Wheeler-Johnson Test energy, provide storage of energy (e.g. starch and
The Wheeler-Johnson test detects the presence of glycogen), important cellular component (e.g.
cytosine and uracil and their derivatives. It does not carbohydrate chain in cell membrane), and
detect thymine because of the methyl group present important structural component in organisms (e.g.
in its structure. When the sample is treated with chitin).
bromine water, it forms yellow-colored 5-bromo-6-
hydroxyhydro derivatives. When barium hydroxide is
added, it produces purple-colored 5,5-dibromo-6-
hydroxyhydro derivatives.

MODULE 6 - CARBOHYDRATES
EXERCISE 9 - QUALITATIVE ANALYSIS OF Figure 6.2. Structure of aldose and ketose.
CARBOHYDRATES
At the end of the exercise, the student should be CLASSIFICATION OF CARBOHYDRATES
able to: Carbohydrates, also known as saccharides or glycans,
are generally divided into four chemical groups
Compare the different qualitative chemical test for according to the number of carbons.
carbohydrates. 1. Monosaccharides – made up of one sugar
Explain the principle involved in each chemical test unit
for carbohydrates.  Hydroxyacetaldehyde (2-carbon sugar)
Interpret the results of a specific chemical analysis of  Glyceraldehyde; Dihydroxyacetone (3-
an unknown carbohydrate sample. carbon sugar)
 Erythose; Erythrulose (4-carbon sugar)
INTRODUCTION TO CARBOHYDRATES  Ribose; Ribulose; Xylose; Xylulose (5-carbon
sugar)
 Glucose; Fructose; Galactose; Mannose (6-
carbon sugar)
  Sedoheptulose (7-carbon sugar) linkages which involve bonding a carbohydrate
 Neuraminic acid (9-carbon sugar) (sugar) molecule to another one, and hence there is
2. Disaccharides – made up of two no reducing group on the sugar; like in the case of
monosaccharide units joined by glycosidic sucrose, glycogen, starch and dextrin.
linkage
 Sucrose (glucose + fructose) MATERIALS, APPARATUS, AND REAGENTS
 Lactose (glucose + galactose) The following are the set of test samples, materials,
 Maltose (glucose + glucose) and reagents used for the qualitative analysis of
 Lactulose (fructose + galactose) carbohydrates.
3. Oligosaccharides – short polymers of usually
three to ten monosaccharide units
 Maltotriose (3 glucose units)
 Dextrin (several glucose units)
4. Polysaccharides – long polymers of
monosaccharides
> Homoglycans (made up of only one type of sugar
unit)
 Cellulose (structural polysaccharide in
plants)
 Chitin (structural polysaccharide in animals)
 Starch (storage polysaccharide in plants)
 Hetastarch (water-soluble starch) MOLISCH'S TEST
 Glycogen (storage polysaccharide in PROCEDURE
animals) 1. Prepare three test tubes and place 20 drops
 Inulin (polyfructan of fructofuranose) of the following sample and label
 Dextran (homopolyglucan of α–1,6 bond) accordingly:
> Heteroglycans (made up of more than one type of 1% glucose
sugar unit, usually a repeating disaccharide units) 1% fructose
 Hyaluronic acid (present in vitreous humor 1% starch
and synovial fluid) 2. To all the test tubes, add 2 drops of Molisch
 Chondroitin sulfate (present in cartilage, reagent.
tendons and ligaments) 3. Then pour 20 drops of concentrated sulfuric
 Dermatan sulfate (present in skin) acid down the side of the test tube (slant
 Keratan sulfate (present in nails) the tube before adding the acid), then erect
 Heparan sulfate (an anticoagulant) the test tube slowly.
 Agarose (found in seaweeds) 4. Observe the color at the junction between
 Peptidoglycan (present in bacterial cell the two layers. Record results.
wall.) 5. Positive result: purple ring at the junction of
the two liquids
Carbohydrates and their derivatives include many PRINCIPLE
other important biomolecules that play key roles in general test for carbohydrates
the immune system, fertilization, preventing carbohydrates when treated with concentrated
pathogenesis, blood clotting and development. sulfuric acid undergo dehydration to give furfural
derivatives
QUALITATIVE CHEMICAL TESTS FOR these compounds condense with α-naphthol to form
CARBOHYDRATES colored products
The following are the qualitative chemical tests for pentoses yield furfural while hexoses yield 5-
the analysis of carbohydrates. Most the chemical hydroxymethylfurfural
tests rely on the interaction of a reducing sugar. A this is a sensitive but a non-specific test and is given
reducing sugar is any sugar that, in a solution, has an positive by all types of carbohydrates
aldehyde or a ketone group. The enolization of reagent: α-naphthol in 95% ethanol
sugars under alkaline conditions is an important (+) result: an appearance of reddish violet or purple
consideration in reduction tests. The ability of a colored ring at the junction of two liquids
sugar to reduce alkaline test reagents depends on
the availability of an aldehyde or keto group for
reduction reactions. Several sugars especially
disaccharides or polysaccharides have glycosidic
 PROCEDURE
Prepare three test tubes and place 20 drops of the
following sample and label accordingly:
1% galactose
1% lactose
1% starch
To all the test tubes, add 20 drops of Benedict’s
reagent.
Figure 6.3. Positive result for Molisch's test (glucose, Boil all the samples in a water bath for 2 minutes.
starch, fructose, galactose). Remove from heat and allow to cool.
Observe for the formation of colored solution and/or
FEHLING'S TEST precipitate. Record results.
PROCEDURE Positive result: green to yellow to orange to red
Prepare a mixture of 2 mL Fehling’s A and 2 mL precipitate (intensity may vary)
Fehling’s B in a test tube. PRINCIPLE
Divide the Fehling's reagent into three test tubes test for reducing sugars
with 1.0 mL each. Label one test tube as control reducing sugars under alkaline conditions
solution. tautomerise and form enediols (powerful reducing
To the remaining two test tubes, place the 2.0 mL of agents)
the indicated sample and label it individually: they reduce cupric ions to cuprous form and are
1% sucrose themselves converted to sugar acids
1% fructose the cuprous ions combine with OH- ions to form
Shake the test tube and boil all the solutions in a hot yellow cuprous hydroxide which upon heating is
water bath for 2 minutes. converted to red cuprous oxide
Observe for the formation of a colored precipitate. it is a semi-quantitative test; the color of the
Compare the results with the control solution. precipitate gives a rough estimate of a reducing
Positive result: brick-red precipitate sugar present in the sample
PRINCIPLE interpretation:
 test for reducing sugars green color - up to 0.5 G% (+)
 carbohydrates with free aldehyde or ketone green precipitate - 0.5-1.0 G% (++)
groups have the ability to reduce solutions yellow precipitate - 1.0-1.5 G% (+++)
of various metallic ions orange precipitate - 1.5-2.0 G% (++++)
 the presence of aldehydes but not ketones is brick red precipitate - >2.0 G% (+++++)
detected by reduction of the deep blue
solution of copper (II) to a red precipitate of
insoluble cuprous oxide [copper (I) oxide]
 the principle involves the reduction of the
cupric ion complexed with tartrate ion to
cuprous oxide
 reagent: Fehling's A [blue aqueous solution
Figure 6.5. Positive result for Benedict's test
of copper (II) sulfate pentahydrate] &
(fructose). Negative result with sucrose and starch.
Fehling's B [clear solution of aqueous potassium
sodium tartrate (Rochelle salt) and a strong alkali
BARFOED'S TEST
(NaOH or KOH)]
PROCEDURE
(+) result: brick red precipitate
Prepare three test tubes and place 20 drops of the
following sample and label accordingly:
1% xylose
1% lactose
1% glycogen
To all the test tubes, add 20 drops of Barfoed’s
reagent.
Boil all the samples in a water bath for 3 minutes.
Remove from heat and allow to cool.
Figure 6.4. Positive result for Fehling's test (fructose). Observe for the formation of colored precipitate.
Negative result with sucrose (non-reducing sugar). Record results.
Positive result: scanty brick-red precipitate
BENEDICT'S TEST PRINCIPLE
test for reducing monosaccharides (+) result: cherry red color
aldoses and ketoses can reduce cupric ions even in
acidic conditions
this test is used to distinguish reducing
monosaccharides from disaccharides by controlling
pH and time of heating
monosaccharides react very fast whereas
disaccharides react very slowly
(+) result: scanty brick red precipitate

Figure 6.8. Positive result for Seliwanoff's test

(sucrose & fructose). Negative result with xylose &
glucose.

BIAL'S TEST
PROCEDURE
Prepare four test tubes and place 20 drops of the
following sample and label accordingly:
Figure 6.6. Positive result for Barfoed's test (xylose & 1% xylose
glucose). Negative result with glycogen. 1% ribose
1% galactose
1% sucrose
To all the test tubes, add 20 drops of Bial’s reagent.
Boil all the samples in a water bath for 2 minutes.
Remove from heat and set aside to cool.
Observe for the change in color of the solution.
Record results.
Positive result: blue-green color of the solution (for
pentoses); brown or red color of the solution (for
Figure 6.7. The scanty brick red precipitate in hexoses)
Barfoed's test.
PRINCIPLE
SELIWANOFF'S TEST test for pentoses
PROCEDURE the test reagent dehydrates pentoses to form
Prepare four test tubes and place 20 drops of the furfural
following sample and label accordingly: furfural further reacts with orcinol and the iron ion
1% xylose present in the test reagent
1% fructose reagent: ferric chloride in orcinol-HCl solution
1% sucrose (+) result: formation of bluish product (all other
1% glucose colors indicate a negative result for pentoses)
To all the test tubes, add 20 drops of Seliwanoff’s hexoses generally react to form green, red or brown
reagent. products
Boil all the samples in a water bath for 2 minutes.
Observe for the change in color of the solution
within 30 seconds upon heating. Record results.
Positive result: cherry red color of the solution
(faster for ketohexoses)

PRINCIPLE
test for ketohexoses
ketohexoses on treatment with hydrochloric acid
form 5-hydroxymethylfurfural which on condensates Figure 6.9. Positive result for Bial's test (xylose &
with resorcinol ribose). Negative result with sucrose, glucose &
this test is given positive by ketohexoses so it is glycogen.
answered by fructose, sucrose and other fructose-
containing carbohydrates HYDROLYSIS TEST
this test distinguishes between glucose and fructose
PROCEDURE a solution of reducing sugar when heated with
Prepare two test tubes and place 40 drops of 1% phenylhydrazine, characteristic yellow crystalline
sucrose on both test tubes. Label one test tube as compounds called osazones are formed
“control” and the other one as “inverted sucrose”. these crystals have definite crystalline structure,
On the “inverted sucrose” test tube, add 3 drops of precipitation time and melting point for different
concentrated hydrochloric acid. reducing sugars
Boil both test tubes in a water bath for 5 minutes. reagent: phenylhydrazine-HCl
Remove from heat and set aside to cool. (+) results: yellow to pale orange crystals with
Add 0.1 M sodium hydroxide dropwise to both specific shape and formation
solutions until the red litmus paper turns to blue.
Perform the Fehling’s test to determine presence of
reducing sugars.
Observe for the formation of precipitate. Record
results.
Positive result: brick-red precipitate
PRINCIPLE Figure 6.11. Positive result for Phenylhydrazine test
sucrose, being a disaccharide, is composed of (formation of osazone crystals specific for each
glucose and fructose units sugar).
inverted sugar is a mixture of glucose and fructose: it
is obtained by splitting sucrose into these two IODINE TEST
components PROCEDURE
the reaction is an acid hydrolysis, where these two Prepare two test tubes and place 40 drops of the
sugar components of sucrose are split following sample and label accordingly:
upon acid hydrolysis, the solution is then tested for 1% starch
the presence of reducing sugars using Fehling’s test 1% glycogen
(+) result of brick-red precipitate indicates successful To both test tubes, add 2 drops of Iodine TS.
hydrolysis of sucrose Observe for changes in the color of the solution.
Record results.
Positive result: blue-violet color for starch; reddish-
brown for glycogen

PRINCIPLE
test for polysaccharides
iodine forms a coordinate complex between the
helically coiled polysaccharide chain and iodine
centrally located within the helix due to adsorption
Figure 6.10. Positive result for Hydrolysis test the color obtained depends upon the length of the
(hydrolysis of sucrose). unbranched or linear chain available for complex
formation
PHENYLHYDRAZINE TEST interpretation:
PROCEDURE Amylose - a linear chain component of starch; gives a
Prepare four test tubes and place 40 drops of the deep blue color
following sample and label accordingly: Amylopectin - a branched chain component of
1% glucose starch; gives a purple color
1% fructose Glycogen - gives a reddish brown color
1% galactose Dextrins - amylo, eryhthro and achrodextrins,
1% lactose formed as intermediates during hydrolysis of starch
To all the test tubes, add 0.2 g of phenylhydrazine give violet, red and no color with iodine respectively
hydrochloride, 0.1 g of sodium acetate, and 5 drops
of glacial acetic acid.
Boil all the samples in a water bath for 5 minutes.
Remove from heat and set aside to cool.
Upon cooling, solid crystals will form. Observe for
the shape of the crystals formed under HPO. Record
results.
PRINCIPLE
osazone crystal formation
Figure 6.12. Positive result for Iodine test with starch • mRNA
(dark blue/violet precipitate).
This enzyme joins the mRNA nucleotides together.
• RNA polymerase (correct answer, your
Which process joins amino acids together to form response)
proteins? • DNA polymerase
• Replication • helicase
• Translation (correct answer, your response)
• Transcription This RNA is a long, single strand of nucleotides.
• mRNA (correct answer, your response)
Which process makes a copy of DNA? • tRNA
• Replication (correct answer, your response) • rRNA
• Translation
• Transcription This RNA is made of two subunits, one large and one
small.
Which process makes mRNA? • mRNA
• Replication • tRNA
• Translation • rRNA (correct answer, your response)
• Transcription (correct answer, your
response) This RNA is a short coiled strand that carries an
anticodon.
Which process occurs at the ribosomes? • mRNA
• Replication • tRNA (correct answer, your response)
• Translation (correct answer) • rRNA
• Transcription (your response)
These nitrogen bases have a two ringed structure.
This is called messenger RNA.
• tRNA • pyrimidines
• rRNA • purines (correct answer, your response)
• mRNA (correct answer, your response)
The structure made of a phosphate, sugar, and a
This is called ribosomal RNA. base is called a
• tRNA • codon
• rRNA (correct answer, your response) • anticodon
• mRNA • nitrogen base
• nucleotide (correct answer, your response)
This is called transfer RNA. • purine
• tRNA (correct answer, your response)
• rRNA Which scientist worked with Francis Crick to propose
• mRNA the structure of DNA?
• Rosalind Franklin
This RNA carries amino acids to the ribosome. • Erin Chargaff
• tRNA (correct answer, your response) • James Watson (correct answer, your
• rRNA response)
• mRNA
Which scientist conducted an x-ray diffraction of
This RNA carries the code from DNA to the ribosome. crystals of purified DNA?
• tRNA
• rRNA
• mRNA (correct answer, your response) • Rosalind Franklin (correct answer, your
response)
Points earned: 1 out of 1 • Erin Chargaff
• James Watson
This RNA joins the amino acids together at the • Francis Crick
ribosome.
• tRNA Which scientist measured the amounts of each
• rRNA (correct answer, your response) nitrogen base found in different species?
• Rosalind Franklin The gene mutation in which a single base is deleted
• Erin Chargaff (correct answer, your or added is called_____________________.
response) • Chromosomal mutation
• James Watson • frameshift mutation (correct answer, your
• Francis Crick response)
• point mutation
RNA contains which sugar? • Chromosomal inversion
• Deoxyribose • Chromosomal translocation
• Ribose (correct answer, your response) • Chromosomal deletion
Choose the base that does not belong in RNA. When a part of a chromosome is cut and then
• adenine reinserted backwards, a __________________
• guanine mutation results.
• thymine (correct answer, your response) • Chromosomal mutation (your response)
• uracil • frameshift mutation
• cytosine • point mutation
• Chromosomal inversion (correct answer)
According to Chargaff's Rule only one of the • Chromosomal translocation
following is true. • Chromosomal deletion
• G=T When part of a chromosome is transferred from one
• A=C chromosome to a non homologous chromosome a
• G=C (correct answer, your response) _______ mutation results.
• C=T • Chromosomal mutation
• A=G • frameshift mutation
• T=U • point mutation
Which kind of bond occurs between the bases in • Chromosomal inversion
DNA? • Chromosomal translocation (correct
• ionic answer, your response)
• covalent • Chromosomal deletion
• nitrogen When part of a chromosome is broken or lost durng
• hydrogen (correct answer, your response) mitosis or meiosis a ___________ mutation results.
• peptide • Chromosomal mutation
What do the phosphate groups join together in • frameshift mutation
DNA? • point mutation
• nitrogen bases • Chromosomal inversion
• sugar molecules (correct answer) • Chromosomal translocation
• nucleotides (your response) • Chromosomal deletion (correct answer,
• codons your response)
There is a double bond in which base pair? The base sequence of DNA is ATA GCA TCC. The
• G-C sequence of RNA transcribed from this strand is
• A-T (correct answer, your response) • ATA GCA TCC
• T-G • CCT ACG ATA
• A-C • CCU ACG AUA
Which base pair is correct? • UAU CGU AGG (correct answer, your
• adenine and cytosine response)
• guanine and cytosine (correct answer, your An mRNA base sequence is UUA GCA. The two
response) anticodons complementary to this are:
• thymine and guanine • AAT CGT
• thymine and cytosine • AAU CGU (correct answer, your response)
The gene mutation in which a single base pair is • TTA GCA
changed in a gene is called____________. • UUA GCA
• Chromosomal mutation One strand of DNA is ATA GCA TCC. What would be
• frameshift mutation the sequence of the complementary strand during
• point mutation (correct answer, your replication?
response) • UAU CGU AGG
• Chromosomal inversion • TAT CGT AGG (correct answer, your
• Chromosomal translocation response)
• Chromosomal deletion • CCT ACG ATA
• CCU ACG AUA Identify the location in the human body where
The rungs of the DNA ladder are protein digestion occurs.
• paired sugars EXERCISE 12 - METABOLISM OF FATS
• paired nucleotides At the end of the exercise, the student should be
• paired nitrogen bases (correct answer, your able to:
response) Describe the process of lipogenesis.
• sugar and phosphate molecules Explain the different metabolic reactions involving
Sides of the DNA ladder are lipids.
• phosphate and base Identify the location in the human body where lipid
• sugar and phosphate (correct answer, your digestion occurs.
response)
• base and phosphate EXERCISE 10 LEARNING TASK
• base and sugar LEARNING TASK
The nucleic acid that is single stranded is____. For the learning task of this module, you need to
• DNA perform a home-based experiment designed to
• RNA (correct answer, your response) extract starch from a plant source. For this
The nucleic acid that remains in the nucleus is____. experiment, you will be utilizing materials available
• DNA (correct answer, your response) at your home, and your resourcefulness as a
• RNA pharmacy student.
The nucleic acid that has three different forms is___.
• DNA As a reminder, always practice safety when
• RNA (correct answer, your response) performing experiments even at home. Wear your
The x-ray diffraction of DNA offered information personal protective equipment (PPE) when
about the DNA molecule's performing this experiment.

MATERIALS, APPARATUS, AND REAGENTS

• base content Test Samples
• shape (correct answer, your response) Starch sources (raw, medium-sized): Potato, Cassava, Sweet
potato
• length
• sugar content Materials & Apparatus
Grater
The sequence of three bases on mRNA is called Tablespoon
a(an)_____. Teaspoon
• anticodon Cheesecloth or filter cloth
Clean glass container
• codon (correct answer, your response)
Reagent
• nucleotide
Distilled or mineral water
• gene 70% Ethyl alcohol
MODULE 7 - METABOLISM Povidone-iodine solution
EXERCISE 10 - EXTRACTION OF STARCH AND PROCEDURES
METABOLISM OF CARBOHYDRATES 1. Starch Extraction from Plant Source
At the end of the exercise, the student should be 2. Choose among the given plant source.
able to: Prepare the plant source of starch by
Extract starch from different plant sources. peeling and grating it using a grater. Take
Identify the properties of starch through physical extra caution when grating the sample.
and chemical analysis. 3. To the grated sample, add one cup of
Describe the conversion of carbohydrates to energy. distilled/mineral water. Make sure to soak
Explain the various metabolic pathways involving all the grated sample in to the water. Press
carbohydrates. the grated sample to extract the starch
Identify the location in the human body where from it.
carbohydrate hydrolysis occurs. 4. Strain the homogenized pulp through a
cheesecloth/filter cloth. Express the
EXERCISE 11 - METABOLISM OF PROTEINS homogenized sample completely. Collect
At the end of the exercise, the student should be the filtrate in a glass container.
able to: 5. Transfer the grated sample into another
Describe the process of protein digestion. container and add one cup of
Explain the various metabolic pathways involving distilled/mineral water. Press the grated
proteins and amino acids. sample to extract the starch from it.
 6. Strain the homogenized pulp through a digested and broken down for the body's metabolic
cheesecloth/filter cloth. Express the needs.
homogenized sample completely. Collect STARCH EXTRACTION
the filtrate and combine it to the previous Polysaccharides are long polymer chains of
filtrate in a glass container. monosaccharides linked by glycosidic linkages. They
7. Allow the starch to settle at the bottom of are quite heterogenous, and have different
the glass container for an hour, and then properties depending on the length of their chain
decant the clear supernatant liquid. and the sugar units they possess. One of the most
8. Add distilled/mineral water to the starch common examples of polysaccharide is starch.
remaining on the glass container, stir, and Starch has many applications, such as in commercial
allow the starch to settle once again for 15 industry to manufacture biofuel, in food industry as
minutes. thickening agent, and in pharmaceutical industry as
9. Decant the clear supernatant liquid once diluent, disintegrant, glidant and binder.
again to collect the starch powder at the
bottom of the glass container. Starch is a homopolymer of glucose forming an α-
glucosidic chain, as shown in Figure 7.13. Starch is a
Identification Tests for Starch storage polysaccharide in plants made up of amylose
Physical Test (about 15-20%) and amylopectin (about 80-85%).
Organoleptic: Determine the color, odor, Table 5.1 summarizes the properties of the two
and appearance of the extracted starch. components of starch.
Solubility: Put a pinch of extracted starch in
a small container and add 1 teaspoon of
distilled water. In another small container,
put a pinch of extracted starch and add 1
teaspoon of ethyl alcohol. Note the
solubility of extracted starch in water and
alcohol and document the result.
Reaction with heat: In a small container,
place a pinch of extracted starch then add 1
teaspoon of distilled water. Then get 1
tablespoon of hot distilled water. Mix both
and observe the product formed. Record its
appearance and consistency and document Figure 7.1. Structure of amylose and amylopectin,
the result. the two components of starch.
Chemical Test
Iodine Test Starch can be sourced from different plant samples.
In a small container, place a pinch of the extracted The most common are: corn (Zea mays), potato
starch then add 1 teaspoon of hot distilled water. (Solanum tuberosum), rice (Oryza sativa), wheat
Using a teaspoon, stir the solution then add 3 drops (Triticum aestivum), arrowroot (Maranta
of povidone-iodine solution. arundinacea), oats (Avena sativa), sweet potato
Observe for any changes in the color of the solution. (Ipomoea batatas), cassava (Manihot esculenta),
Record, document and interpret the result. green pea (Pisum sativum).
PROPERTIES AMYLOSE AMYLOPECTIN
STARCH EXTRACTION linearity linear, helical branched (α-
(α-1,4 bond) 1,4 & α-1,6)
solubility in insoluble soluble
water
size 250-300 units >1000 units
iodine test dark blue blue-violet

enzyme amylase α-glucosidase

& dextrinase

Table 7.1. Properties of amylose and amylopectin.

The body converts complex carbohydrates into Starch becomes soluble in water when heated. The
utilizable form of energy from our dietary intake. starch granules swell, and small amylose molecules
Through hydrolysis, these carbohydrates are
leaches out and forms a network that holds the 1. To each of the four test tubes, add 3 drops
water which increases the viscosity of the mixture. of iodine TS.
This process is termed as starch gelatinization. This 2. Observe for any changes in the color of the
property enables it to be used as thickening agent in solution.
cooking or as a paste due to its viscosity. The most Benedict's Test
common test to identify starch is the iodine test. The 1. To each of the four test tubes, add 3.0 mL of
iodine TS is composed of an iodine dissolved in Benedict's reagent.
potassium iodide, which forms a triiodide ion. This 2. Place all the test tubes in a boiling water
ion forms a complex with the starch which results to bath for 5 minutes.
formation of blue to violet color of the solution. The 3. Observe for any changes in the color of the
intensity of the color depends on the amount of solution.
starch molecules present in a solution. When viewed
under the microscope, starch granules tend to have DIGESTION OF CARBOHYDRATES
an angular outline and some of them are attached Carbohydrate digestion are biochemical processes by
with each other to form larger granules. which carbohydrates from food are broken down
into simpler chemical units by hydrolysis that can be
METABOLISM OF CARBOHYDRATE used by cells for their metabolic needs. Digestion is
PROCEDURE OF EXPERIMENT the first stage in the processing of food products.
Below is the basic procedure to demonstrate
carbohydrate metabolism. Read and analyze the
procedures carefully.

Test Samples Materials & Apparatus Reagent

5% Starch 5 mL Pipet 5% Hydrochloric acid
Beaker Benedict’s reagent
Bulb aspirator Distilled water
Glass droppers Iodine TS
Glass rod Saliva
Hot plate
Test tubes
Thermometer

PROCEDURES
Hydrolysis of Starch
1. Let about 5.0 mL saliva drop from your
lower lip into a beaker. Do not make any
Figure 7.2. Digestion of carbohydrates.
chewing motions while doing so, otherwise
The figure above shows the summary on how the
the saliva gets frothy.
body digest dietary carbohydrates. As previously
2. Dilute the saliva with an equal amount of
discussed, carbohydrates could be monosaccharide,
distilled water. This will serve as the
disaccharide, oligosaccharide or polysaccharide.
amylase solution.
These carbohydrates are digested and hydrolyzed
3. Prepare four test tubes and label them 1 to
through a series of enzymatic reactions, starting
4. Add the following solutions to the four
from the mouth down the intestines, until it is
test tubes:
absorbed into the bloodstream.
4. 1 = 3.0 mL of distilled water
5. 2 = 3.0 mL amylase solution
CARBOHYDRATE DIGESTION PROCESS IN THE
6. 3 = 3.0 mL amylase solution + 10 drops 5%
TONGUE
hydrochloric acid
Fast-releasing carbohydrates stimulate the
7. 4 = 3.0 mL amylase solution + place in
sweetness taste sensation, which is the most
boiling water bath for 5 minutes
sensitive of all taste sensations. Even extremely low
8. To all the four test tubes, add 5.0 mL of 5%
concentrations of sugars in foods will stimulate the
starch.
sweetness taste sensation. Sweetness varies
9. Place all the test tubes in a 37°C water bath
between the different carbohydrate types—some
for 1 hour.
are much sweeter than others. Fructose is the top
10. After the incubation, split the four
naturally occurring sugar in sweetness value. Less-
incubated solutions equally into two sets of
sweet whole grains (starches and fiber) take longer
small test tubes. One set of solution will be
to chew and get sweeter the more you chew them.
used for Iodine test, whereas the second set
Once in the stomach, whole-grain food takes longer
will be used for Benedict's test.
to digest and keep you full longer.
Iodine Test
 pancreatic juice through a duct. The pancreatic juice
contains pancreatic amylase, which starts again the
breakdown of dextrin into shorter and shorter
carbohydrate chains.

Figure 7.4. Digestion of carbohydrates at the

Figure 7.3. Taste sensations in the tongue.
intestines.
Additionally, enzymes are secreted by the intestinal
FROM MOUTH TO STOMACH
cells that line the villi. These enzymes are sucrase,
When the dietary carbohydrates reach the stomach
maltase, and lactase. From their respective names,
no further chemical breakdown occurs: the amylase
they hydrolyze specific sugar, particularly a
enzyme does not function in the acidic conditions of
disaccharide:
the stomach. Hence, the mechanical breakdown is
 sucrase - hydrolyze sucrose into glucose and
ongoing – the strong peristaltic contractions of the
fructose
stomach mix the carbohydrates into the more
 maltase - hydrolyze maltose into two
uniform mixture of chyme.
glucose molecules
 lactase - hydrolyze lactose into glucose and
Mechanical digestion Chemical digestion
galactose
chewing or mastication involves salivary
Once carbohydrates are chemically broken down
amylase
into single sugar units they are then transported into
the inside of intestinal cells.
crumbles the breaks the bonds
carbohydrate foods between the INTO THE BLOODSTREAM
into smaller and monomeric sugar units The small intestine cells have membranes that
smaller pieces of disaccharides, contain many transport proteins in order to get the
oligosaccharides, and monosaccharides and other nutrients into the blood
starches where they can be distributed to the rest of the
body. Fructose enters the bloodstream through
breaks down amylose facilitated diffusion, while glucose and galactose
and amylopectin into enters through active transport. The
dextrin and maltose monosaccharides absorbed are then transferred to
the liver where it converts galactose to glucose,
only about 5% of breaks fructose into even smaller carbon-containing
starches are broken units and stores glucose as glycogen or exports it
down in the mouth back to the blood. Always remember that the
(more glucose in the utilizable form of sugar in the body is glucose.
mouth would lead to Fructose and galactose must be converted in order
tooth decay) for the body to utilize them.
Table 7.2. Mechanical and chemical digestion of
carbohydrates.

FROM STOMACH TO SMALL INTESTINES

The chyme formed in the stomach is gradually
expelled into the duodenum. Upon entry of the
chyme into the small intestine, the pancreas releases
 high-fiber foods (i.e. whole grains) are linked to a
decrease in weight gain and reduced risk of chronic
diseases.
EXPERIMENT RESULT (CARBOHYDRATE
METABOLISM)
STARCH HYDROLYSIS
The starch solution was placed into the four test
tubes. After which, each test tube are added with
the following:
 Test tube 1 - starch solution + distilled water
 Test tube 2 - starch solution + saliva
 Test tube 3 - starch solution + saliva + 5% HCl
 Test tube 4 - starch solution + heated saliva (the saliva
was heated prior)
All the samples are incubated at 37°C water bath,
Figure 7.5. Transport of the converted mimicking the body temperature. The goal is to
monosaccharides into the liver. know which among the following will yielded a
hydrolyzed starch. To know the result, all the
How much glucose the liver exports to the blood is samples are subjected to Lugol's and Benedict's
under hormonal control and you will soon discover tests.
that even the glucose itself regulates its
concentrations in the blood.

There are several metabolic processes in the body

that regulates the amount of glucose needed by the
body, whenever the situation requires. There are
metabolic processes triggered when the blood Figure 7.6. Starch solution was placed into the test
glucose levels drops, while there are also processes tube with water and the enzyme amylase.
triggered when the blood glucose levels are too high,
all of these are under hormonal control of the insulin LUGOL'S TEST
and glucagon. After incubation, an aliquot portion from the four
 Glycolysis - converts glucose → pyruvate samples were subjected to Lugol's test or Iodine test.
 Glycogenolysis - converts glycogen → As previously discussed, Iodine test is used to
glucose determine the presence of starch if it will yield a dark
 Glycogenesis - converts glucose → glycogen blue or violet color. Therefore, if the sample yielded
 Gluconeogenesis - converts a non- some dark blue or violet precipitate , it only means
carbohydrate → glucose that it still has unhydrolyzed starch. Hence, after the
incubation period, the starch sample was
LEFTOVER CARBOHYDRATES unsuccessfully hydrolyzed.
Some of the remaining indigestible carbohydrates
(fiber-rich food) are broken down by enzymes After the Lugol's test was performed, the results are
released by bacteria in the large intestine. The shown on Figure 7.7.
products of bacterial digestion of these slow-
releasing carbohydrates are short-chain fatty acids
and some gases (the reason why smelly gases are
released). The short-chain fatty acids are either used
by the bacteria to make energy and grow, are
eliminated in the feces, or are absorbed into cells of
the colon, with a small amount being transported to
the liver. The colonic cells may also use the short-
chain fatty acids to support some of their functions.
While the liver can also metabolize the short-chain
fatty acids into cellular energy.
Figure 7.7. Lugol's test performed on each sample.
Dietary fiber is digested much less in the GI tract
than other carbohydrate types (simple sugars, many Notice that only the sample 2 yielded negative with
starches). The rise in blood glucose after eating them Lugol’s test, the rest of the samples yielded a dark
is less and slower. These physiological attributes of blue precipitate. This only indicates that only Test
tube 2 was successful in hydrolyzing the starch role in energy production, protein metabolism plays
sample after incubation. an important role in maintaining good health.
 Test tube 1 = does not hydrolyze starch
since it is only water PROCEDURE OF THE EXPERIMENT
 Test tube 3 = starch was unhydrolyzed Below is the basic procedure to demonstrate protein
because the HCl solution denaturated the metabolism. Read and analyze the procedures
amylase carefully.
 Test tube 4 = starch was unhydrolyzed
because the heat denaturated the amylase MATERIALS, APPARATUS, AND REAGENTS
BENEDICT'S TEST Test Samples
To confirm if the Test tube 2 was really successful in Boiled egg white
Boiled fish meat
hydrolyzing the starch, Benedict's test was
performed. Remember that in previous discussions,
Materials & Apparatus
Benedict's test detects the presence of reducing Hot plate
sugars, and glucose is one of them. Benedict's test is Ice
semi-quantitative test; the amount of reducing sugar Test tubes
present affects the color change of the solution; Thermometer
5 mL Pipet
brick-red precipitate occurs when there is an Beaker
overwhelming amount of the reducing sugar. Bulb aspirator
Therefore, if there is a change in color, it would only Glass droppers
indicate that the starch solution was hydrolyzed.
Reagent
5% Hydrochloric acid
After the Benedict's test was performed, the results 5% Sodium hydroxide
are shown on Figure 7.8. Distilled water
Pancreatin solution

PROCEDURES
1. Obtain two sets of five big test tubes. Label
the first set for "egg white" while the
second set for "fish"
2. Add a small sliver of hard-boiled egg white
to the first set of five test tubes. Add a small
chunk of boiled fish meat to the second set
of five test tubes.
3. Add the following solutions to the sample
and label them accordingly:
Figure 7.8. Benedict's test performed on each 1 = Add 5 mL pepsin solution and 10 drops
sample. distilled water
2 = Add 5 mL pepsin solution and 10 drops
This time around, notice that only the sample 2 5% HCl
yielded positive with Benedict’s test, and the rest of 3 = Add 5 mL pepsin solution and 10 drops
the samples yielded negative result. This only 5% HCl; place on an ice bath
indicates that only Test tube 2 was successful in 4 = Add 5 mL distilled water and 10 drops
hydrolyzing the starch sample after incubation . 5% HCl
 Test tube 1 = no reducing sugars present 5 = Add 5 mL pepsin solution and 10 drops
(water does not hydrolyze starch) 5% NaOH
 Test tube 3 = no reducing sugars present 4. Place test tubes 1, 2, 4 and 5 in 37°C water
(the denaturated amylase due to HCl bath and incubate for 90 minutes.
solution was unsuccessful in hydrolyzing the 5. Examine all the test tubes after the
starch) incubation. Note for any digestion that have
 Test tube 4 = no reducing sugars present occurred and any changes in the color of
(the denaturated amylase due to heat was the solution.
unsuccessful in hydrolyzing the starch)
METABOLISM OF PROTEINS DIGESTION OF PROTEINS
From an energy production standpoint, proteins The body’s digestive system breaks down the protein
supply only about 10%, while the rest comes from (denatured or not) into the individual amino acids.
fats and carbohydrates. However, despite its minor The amino acids are absorbed and used by cells to
build other proteins and a few other powerful mechanical stomach contractions churn
macromolecules, such as DNA. the partially digested protein into a more uniform
mixture called chyme.

Protein digestion in the stomach takes a longer time

than carbohydrate digestion, but a shorter time than
fat digestion. Eating a high-protein meal increases
the amount of time required to sufficiently break
down the meal in the stomach; the food remains in
the stomach longer, making you feel full longer.

FROM STOMACH TO SMALL INTESTINES

The stomach empties the chyme containing the
broken down egg pieces into the small intestine,
Figure 7.9. Digestion of proteins. where the majority of protein digestion occurs. The
Eggs are a good dietary source of protein and will be pancreas secretes digestive juice that contains more
used as our example to describe the path of proteins enzymes that further break down the protein
in the processes of digestion and absorption. One fragments. The two major pancreatic enzymes that
egg, whether raw, hard-boiled, scrambled, or fried, digest proteins are chymotrypsin and trypsin. The
supplies about 6 g of protein. cells that line the small intestine release additional
PROTEIN DIGESTION PROCESS enzymes that finally break apart the smaller protein
IN THE MOUTH fragments into the individual amino acids. The
Unless you are eating it raw, the first step in egg muscle contractions of the small intestine mix and
digestion (or any other protein food) involves propel the digested proteins to the absorption sites.
chewing. The teeth begin the mechanical breakdown The goal of the digestive process is to break the
of the large egg pieces into smaller pieces that can protein into dipeptides and amino acids for
be swallowed. The salivary glands provide some absorption.
saliva to aid swallowing and the passage of the
partially mashed egg through the esophagus. The
mashed egg pieces enter the stomach through the
esophageal sphincter.

FROM MOUTH TO STOMACH

The stomach releases gastric juices containing HCl
and pepsin, which initiate the breakdown of the
protein. The acidity of the stomach facilitates the
unfolding of the proteins that still retain part of their
3D structure after cooking and helps break down the
protein aggregates formed during cooking.

Figure 7.11. The digestion of proteins involves

several enzymatic reactions.
All the enzymes involved in protein digestion are
proteolytic, breaking the long chainlike molecules of
Figure 7.10. Chemical and mechanical digestion of
proteins into shorter fragments (peptides) and
proteins in the stomach.
eventually into their components, amino acids.
pancreatic juice:
Pepsin, secreted by the chief cells of stomach,
dismantles the protein chains into smaller and  trypsin
smaller fragments, while HCl secreted by the parietal  chymotrypsin
cells of the stomach, also helps in protein digestion.  carboxypeptidase
intestinal mucosal cells:
Egg proteins are large globular molecules and their  aminopeptidase
chemical breakdown requires time and mixing. The At the distal duodenum-proximal jejunum, amino
acids are transported from intestinal lumen to the
blood, which requires active transport and ATP to
proceed. Once the amino acids are in the blood, they
are transported to the liver (which acts as checkpoint
for amino acid distribution and any further
breakdown).

More than 90% of the protein ingested does not get

broken down further than the amino acid monomers
and very little protein makes it to the large intestine Figure 7.12. Starch solution was placed into the test
if you are not eating excessive amounts. If you have tube with water and the enzyme amylase.
smelly flatulence, this may be a sign you are eating
too much protein because the excess is making it to The degree of hydrolysis is observed based on the
the colon where you gut microbes are digesting it change in the appearance of the sample and the
and producing smelly gas. turbidity in the test tube.

PROTEIN ABSORPTION METABOLISM OF FATS

In adults, essentially all protein is absorbed as
tripeptides, dipeptides or amino acids and this
process occurs in the duodenum or proximal
jejunum of the small intestine. The peptides and/or
amino acids pass through the interstitial brush
border by facilitative diffusion or active transport.
Active transport sodium and ATP actively transport
the molecule through the cell membrane. The R High-fat food remains in the stomach longer than
group determines the type of transporter used. Once those low in fat and the conversion of high-fat
passed through the membrane, the amino acids or materials into chyme takes longer. This is why high-
peptides are released into the intestinal blood fat meal causes a person to feel full for a longer
stream and are transported to the liver by the period of time.
hepatic (liver) portal vein. This is known as the
enterohepatic circulation. PROCEDURE OF THE EXPERIMENT
Below is the basic procedure to demonstrate lipid or
In the liver, 50-65% remain and are used to fat metabolism. Read and analyze the procedures
synthesize protein, nitrogen containing compounds carefully.
and purine/pyrimidine bases. In some cases, they MATERIALS, APPARATUS, AND REAGENTS
may be converted to energy. The liver regulates the Test Samples Materials & Apparatus Reagent
amino acid levels in the blood. The amino acids that Heavy cream 5 mL Pipet 5% Hydrochloric acid
do not stay in the liver pass through and are Beaker Animal bile
Bulb aspirator Distilled water
transported to the rest of the body to be taken up
Hot plate Pancreatin solution
and utilized by other cells. Most branch chain amino Paper towels
acids pass through the liver. pH meter
Test tubes
Thermometer
EXPERIMENT RESULT (PROTEIN METABOLISM)
Wash bottle
EGG AND FISH MEAT HYDROLYSIS PROCEDURES
There are two sets of prepared test tubes: 5 test 1. Obtain a set of three test tubes.
tubes for egg and 5 test tubes for fish meat. Both are 2. Add 3.0 mL of heavy cream to each test
added with the following reagents: tube.
3. Add the following solutions to each test
Test tube 1 = pepsin + distilled water tube and label them accordingly:
Test tube 2 = pepsin + 5% HCl 1 = Add 5.0 mL distilled water and 1.0 mL
Test tube 3 = pepsin + 5% HCl (the sample is placed bile solution
in an iced bath) 2 = Add 5.0 mL pancreatin solution
Test tube 4 = distilled water + 5% HCl 3 = Add 5.0 mL pancreatin solution and 1.0
Test tube 5 = pepsin + 5% NaOH mL bile solution
All the samples, except test tube 3, are incubated at 4. Shake all the test tubes vigorously to mix all
37°C water bath, mimicking the body temperature, the contents.
thereby inducing reaction. 5. Measure the initial pH of each solution
using pH meter.
 6. Place all the test tubes into 37°C water bath churning and contractions help to disperse the fat
for 20 minutes. molecules, while the diglycerides derived in this
7. Measure again the pH of the solutions. process act as further emulsifiers. However, amid all
8. Place again the samples into the same of this activity, very little fat digestion occurs in the
water bath for 20 minutes more and stomach.
measure the pH once again.
9. Place again the samples into the same
water bath for 20 minutes more and
measure the pH for the last time.
DIGESTION OF LIPIDS
Like carbohydrates and protein, lipids are broken
into small components for absorption. Since most of
our digestive enzymes are water-based, certain
processes are peculiar to lipid digestion only.

Figure 7.14. Chemical and mechanical digestion of

lipids in the stomach.

FROM STOMACH TO SMALL INTESTINES

Figure 7.13. Digestion of lipids. As stomach contents enter the small intestine, the
The figure above summarizes the enzymatic digestive system sets out to manage a small hurdle,
reactions involved in digesting dietary lipids. namely, to combine the separated fats with its own
However, compared to carbohydrate and proteins, watery fluids. The solution to this hurdle is bile. Bile
lipids take much longer time to digest. contains bile salts, lecithin, and substances derived
from cholesterol so it acts as an emulsifier. It attracts
LIPID DIGESTION PROCESS and holds on to fat while it is simultaneously
IN THE MOUTH attracted to and held on to by water. Emulsification
The first step in the digestion of triacylglycerols and increases the surface area of lipids over a thousand-
phospholipids begins in the mouth as lipids fold, making them more accessible to the digestive
encounter saliva. Next, the physical action of enzymes.
chewing coupled with the action of emulsifiers
enables the digestive enzymes to do their tasks. The Once the stomach contents have been emulsified,
enzyme lingual lipase, along with a small amount of fat-breaking enzymes work on the triacylglycerols
phospholipid as an emulsifier, initiates the process of and diglycerides to sever fatty acids from their
digestion. These actions cause the fats to become glycerol foundations. As pancreatic lipase enters the
more accessible to the digestive enzymes. As a small intestine, it breaks down the fats into free fatty
result, the fats become tiny droplets and separate acids and monoglycerides. Yet again, another hurdle
from the watery components. presents itself. How will the fats pass through the
watery layer of mucous that coats the absorptive
FROM MOUTH TO STOMACH lining of the digestive tract? As before, the answer is
In stomach, gastric lipase starts to break down bile. Bile salts envelop the fatty acids and
triacylglycerols into diglycerides and fatty acids. monoglycerides to form micelles.
Within 2-4 hours after eating a meal, roughly 30% of
the triacylglycerols are converted. The stomach's
 INTO THE BLOODSTREAM result from diseases that affect absorption, such as
Micelles have a fatty acid core with a water-soluble Crohn’s disease and cystic fibrosis.
exterior. This allows efficient transportation to the
intestinal microvillus. Here, the fat components are EXPERIMENT RESULT (FAT METABOLISM)
released and disseminated into the cells of the HYDROLYSIS OF FAT COMPONENTS FROM HEAVY
digestive tract lining. CREAM
Three test tubes are prepared and placed with heavy
cream as the source of fat. Then the following
regaents are added to the sample

Test tube 1 = distilled water + animal bile

Test tube 2 = pancreatin
Test tube 3 = pancreatin + animal bile
Figure 7.15. Micelles allow fats to be transported All the samples, except test tube 3, are incubated at
into the bloodstream. 37°C water bath, mimicking the body temperature,
thereby inducing reaction.
Inside the intestinal cells, the monoglycerides and
fatty acids reassemble themselves into
triacylglycerols. Triacylglycerols, cholesterol, and
phospholipids form lipoproteins when joined with a
protein carrier. Lipoproteins have an inner core that
is primarily made up of triacylglycerols and
cholesterol esters (a cholesterol ester is a cholesterol
linked to a fatty acid). The outer envelope is made of
phospholipids interspersed with proteins and Figure 7.17. The resultant samples after adding the
cholesterol. Together they form a chylomicron, respective reagents.
which is a large lipoprotein that now enters the
lymphatic system and will soon be released into the The pH was then measured to detect acidity caused
bloodstream via the jugular vein in the neck. by the hydrolysis of the fat present in the cream into
Chylomicrons transport food fats perfectly through glycerol and fatty acids.
the body’s water-based environment to specific
destinations such as the liver and other body tissues.

Figure 7.18. The

samples during the
Figure 7.16. pH measurement.
Chylomicrons
are large
lipoproteins
that transports
and carries
dietary fats.

Cholesterols are poorly absorbed when compared to

phospholipids and triacylglycerols. Cholesterol MODULE 8 - BLOOD AND URINE ANALYSIS
absorption is aided by an increase in dietary fat
components and is hindered by high fiber content. EXERCISE 13 - BLOOD CHEMISTRY
This is the reason that a high intake of fiber is At the end of the exercise, the student should
recommended to decrease blood cholesterol. Foods
be able to:
high in fiber such as fresh fruits, vegetables, and oats
can bind bile salts and cholesterol, preventing their
Identify the different components of blood
absorption and carrying them out of the colon. through qualitative chemical tests.
Analyze the process of examining coagulated
If fats are not absorbed properly as is seen in some and whole blood samples through qualitative
medical conditions, a person’s stool will contain high analysis.
amounts of fat. If fat malabsorption persists the Explain the results and its relevance to the
condition is known as steatorrhea. Steatorrhea can functions of the blood components.
EXERCISE 14 - URINALYSIS 2. After which allow the blood to
At the end of the exercise, the student should coagulate for 15-20 minutes or until a
be able to: clear yellow fluid appears on top of the
Identify the different parameters being checked blood to get the serum. Do not shake
in a routine urinalysis and the significance of the tube to avoid blood hemolysis
each parameter. (indicated by red coloration of the
Analyze the process of examining a sample serum).
urine using urine dipstick. 3. Once the blood has already coagulated,
Explain the results of the urinalysis. gently remove the red cap of the
BLOOD CHEMISTRY vacutainer and centrifuge the blood
sample at 2500 rpm for 5 minutes to
separate the clot from the serum. If the
serum fails to separate from the blood
clot, repeat the centrifugation.
4. If a red colored serum is produced after
centrifugation, discard the sample and
get a new one.
5. Once the serum is separated, pipet out
the serum and equally divide it into four
Certain qualitative tests can be performed to small test tubes.
chemically characterize the different 6. Label each test tubes as follows:
components of blood. Knowing this enables us 1 - Test for Carbohydrates
to fully understand the functions and properties 2 - Test for Proteins
of blood. 3 - Test for Chlorides
4 - Test for Phosphates
PROCEDURE OF THE EXPERIMENT
Below is the procedure to identify the different Coagulated Blood Extraction (Red Cap
chemical components of blood. Read and Vacutainer): Obtaining the Fibrin
analyze the processes carefully. 1. After the serum had been separated
from the clotted blood, break the clot in
MATERIALS, APPARATUS, AND REAGENTS the tube using a glass rod and place it in
Test Samples Materials & Apparatus Reagent
Blood 5 mL Pipet 1% Sodium chloride a filter paper.
5 mL Red cap vacutainer 10% Ammonium hydroxide
5 mL Purple cap vacutainer 10% Nitric acid 2. Press out the remaining serum in the
Beaker
Bulb aspirator
5% Ammonium molybdate
5% Silver nitrate
clot and dry it using the filter paper to
Bunsen burner
Centrifuge
95% Ethanol
Acetic anhydride
obtain the fibrin.
Evaporating dish Ammonium sulfate crystals 3. Wash the obtained fibrin with distilled
Filter paper Benedict’s reagent
Glass droppers Chloroform water using a micropipette by slowly
Glass rod Concentrated sulfuric acid
Hot plate Diluted hydrochloric acid dropping water into it to further
Sealing film Distilled water
Test tubes Hopkins-Cole reagent remove excess serum.
Tripod
Wire gauze
Millon’s reagent
Petroleum ether
4. Dry the fibrin again by pressing filter
Potassium ferricyanide
Saturated ammonium sulfate
paper against it.
Standard cholesterol solution 5. After the fibrin has dried, separate it
Stoke’s reagent

PROCEDURES into two test tubes and label it as

Coagulated Blood Extraction (Red Cap follows:
Vacutainer): Obtaining the Serum 1 - Test for Tryptophan
1. Have a member of the group be 2 - Test for Soluble Proteins
extracted with 5.0 mL of fresh blood Whole Blood Extraction (Purple Cap Vacutainer)
sample. Extraction of blood should be 1. Have another member of the group be
done by a phlebotomist or a senior extracted with 5.0 mL of fresh blood
medical technology student. The blood sample. Extraction of blood should be
sample should be placed in a red cap done by a phlebotomist or a senior
vacutainer (no EDTA/anticoagulant). medical technology student. The blood
 sample should be placed in a purple cap 1. To the fourth portion of the serum,
vacutainer (with EDTA/anticoagulant). acidify with 2 drops of 10% nitric acid,
2. Mix the blood sample inside the then add 2.0 mL 5% ammonium
vacutainer via tumbling method to get molybdate.
the whole blood. 2. Heat the sample in a hot water bath
3. Once the whole blood has been until precipitates form.
obtained, remove the purple cap from 3. Note for the formation of yellow
the vacutainer and take 1.0 mL of the precipitate indicating the presence of
blood and place it in a big test tube and phosphates.
label it as 1 - Test for Cholesterol.
4. Take another 2.0 mL of the whole blood Fibrin Tests
and place it in an evaporating dish and Test for Tryptophan
label it as 2 - Test for Iron. 1. To the first portion of the fibrin, add 3.0
5. Lastly, take 1.0 mL of the whole blood mL Hopkin Cole's reagent in the test
and place it in a big test tube and label tube.
it as 3 - Test for Blood Gases. 2. Add 3.0 mL concentrated sulfuric acid
dropwise, in a slanted test tube, to
Serum Tests prevent rapid reaction.
Test for Carbohydrates 3. Note for the formation of a purple ring
1. To the first portion of the serum, add 5 the junction of two layers of the liquid
drops of Benedict’s reagent. indicating the presence of tryptophan.
2. Heat the sample in a boiling water bath.
3. Note for the formation of a blue colored Test for Soluble Proteins
solution or formation of a brick red 1. To the second portion of the fibrin, add
precipitate. 2.0 mL Millon’s reagent.
2. Heat the sample in a hot water bath for
Test for Proteins 10 minutes.
1. To the second portion of the serum, add 3. Note for the formation of red colored
an equal volume of saturated solution with precipitate indicating the
ammonium sulfate to purify the presence of tyrosine (a common amino
proteins and allow precipitation of acid in proteins) and soluble proteins.
cellular proteins.
2. Filter the solution. Set aside the filtrate, Whole Blood Tests
and then on the residue, add 1% Test for Cholesterol
sodium chloride dropwise until changes 1. Place 11 mL of ethanol-petroleum ether
occur. Note observations. mixture (3:1) in a big test tube then add
3. Then on the filtrate, add ammonium 1.0 mL of the whole blood sample
sulfate crystals until the solution is slowly. The ethanol-petroleum ether
saturated. mixture will separate the water soluble
4. Note for the presence of precipitates from the lipid soluble components of
which indicates the presence of the blood.
albumin and globulin in the blood. 2. Place a sealing film in the test tube and
tumble it for 2 minutes. Allow the test
Test for Chlorides tube to settle for 30 minutes in the rack.
1. To the third portion of the serum, 3. Then transfer the contents of the test
acidify with 2 drops of 10% nitric acid, tube to an evaporating dish and
then add 5 drops 5% silver nitrate. evaporate it to incipient dryness using a
2. Note for the formation of white steam bath. Water soluble components
precipitate indicating the presence of will evaporate, leaving the lipid soluble
chlorides. components in the evaporating dish.
4. After evaporating, extract the residue
Test for Phosphates left with 2.5 mL chloroform. Leave the
 chloroform and residue in the
evaporating dish for 2 minutes, then Test for Blood Gases
transfer the contents in a big test tube. 1. Dilute 1.0 mL of the whole blood
5. Repeat the extraction by adding sample with 3.0 mL of distilled water in
another 2.5 mL chloroform again to the a big test tube. Cover with a sealing film
residue in the evaporating dish, leaving then shake.
it for 2 minutes. 2. Presence of oxyhemoglobin will yield a
6. Transfer the second chloroform extract bright red colored solution. Transfer
to the same big test tube where the half of this solution to another big test
first chloroform extract to make a total tube and label it as control.
of 5 mL chloroform extract. 3. On a different test tube, place 1.0 mL of
7. To that 5 mL chloroform extract, add Stoke’s reagent and 10% ammonium
2.0 mL acetic anhydride and 0.1 mL hydroxide dropwise until precipitates
concentrated sulfuric acid. Place a dissolve. This will act as a reducing
sealing film on the test tube and mix the agent.
contents by tumbling several times. 4. Add a few drops of the prepared
8. Set away the sample in a dark place for reducing agent to the second test tube
15 minutes. containing oxyhemoglobin. Shake the
9. On a separate test tube, measure 5.0 test tube vigorously.
mL standard cholesterol solution (0.2 g 5. The compound formed is reduced
cholesterol powder in 200 mL hemoglobin and its color is usually
chloroform). purple to blue.
10. Add to standard cholesterol solution 2.0
mL acetic anhydride and 0.1 mL sulfuric BLOOD
acid. Blood is a connective tissue, flowing through
11. Place a sealing film on the test tube and capillaries, veins and arteries of all vertebrates.
mix the contents by tumbling several The red color is characteristic due to the
times. Set away the sample in a dark presence of hemoglobin in the red blood cells.
place for 15 minutes. Its main function is the logistics of distribution
12. Compare the intensity of the and systemic integration, whose containment in
chloroform extract to the standard the blood vessels (vascular space) supports its
cholesterol solution. distribution (blood circulation) to almost the
13. A positive result will start as a purplish entire body.
pink color then progresses to a light
green to dark green color of the
solution.

Test for Iron

1. Place 2.0 mL of the whole blood sample
in an evaporating dish and heat over a
Bunsen flame until ash is formed. Do
this inside the fume hood.
2. Cool the evaporating dish and add 5.0 Figure 8.1. Main components of blood.
mL of diluted hydrochloric acid to the Blood is a type of connective tissue specialized
ash residue. with a matrix colloidal liquid and a complex
3. Leave it for about 2 minutes then filter constitution. It is composed of a solid phase
into a big test tube. (formed elements), which includes the
4. Add 10 drops of potassium ferricyanide erythrocytes (or red blood cells), the leukocytes
to the solution. (white blood cells) and platelets, and a liquid
5. Note for the formation of an intense phase, represented by blood plasma. These
blood red color of the solution phases are also called blood components, which
indicating the presence of iron. are divided in serum component (liquid phase)
and cellular component (solid phase). About 7- washed to remove the fibrin (a solid yellow-
8% of the total body weight is blood. An white substance).
average-sized man has about 5.7 L of blood,
while an average-sized woman has 4.3 L of Figure 8.11. Fibrin is isolated from the blood
blood. sample after the serum separated.

Whole blood
The blood sample is placed in a purple cap
vacutainer (contains an anticoagulant, e.g.
EDTA). The whole blood (tumbling) is shaken
through tumbling method to prevent clotting.

Figure 8.12. Whole blood sample.

Figure 8.2. Specific composition of the whole

blood in the body.

QUALITATIVE TESTS FOR BLOOD

BLOOD SAMPLES TESTS FOR BLOOD SERUM
In this exercise, the blood will be tested for its TEST FOR CARBOHYDRATES
components through a series of qualitative The test used Benedict’s reagent to detect the
chemical tests. The procedure will require two presence of reducing sugar. Formation of brick red
blood samples. precipitate is a positive result.
Figure 8.13.
Benedict's test
Coagulated blood
positive result. The
The blood sample is placed in a red cap intensity of the color
vacutainer (do not contain any anticoagulant). depends on the
After the blood is coagulated, it is subjected to amount of sugar
centrifugation, where the serum (a clear yellow present in sample.
solution) separates from the clot, as shown in
Figure 8.10. TEST FOR PROTEINS
The test used ammonium sulfate to detect the
Figure presence of proteins. The presence of precipitates
8.10. indicates the presence of albumin and globulin in the
blood.
Difference
between
blood
plasma and Figure 8.14. Positive result for
blood ammonium sulfate test.
serum.

After the serum TEST FOR CHLORIDES

was separated, The test used silver nitrate to detect the presence of
the clot is isolated. chlorides in blood (around 97-107 mEq/L). The
reaction results to formation of silver chloride which
The clot is then
appears as a white precipitate.
 Figure 8.15. Silver nitrate test
positive result (white
precipitate).
TESTS FOR WHOLE BLOOD
TEST FOR CHOLESTEROL
The test used acetic acid-sulfuric acid to detect the
presence of cholesterol in the blood sample. The
presence of the hydroxyl group on the cholesterol
TEST FOR PHOSPHATES and the unsaturated bonds on the adjacent fused
The test used ammonium molybdate to detect the ring results to formation of green color of the
presence of phosphates in the blood, which is solution.
important for energy production, muscle and nerve
function, bone growth and acid-base balance
reactions. The reaction results to formation of
ammonium phosphomolybdate which appears as Figure 8.19. Positive result for
yellow precipitate. cholesterol test using acetic acid-
sulfuric acid.
Figure 8.16. Ammonium
molybdate test positive result
indicates presence of
phosphate groups.
TEST FOR IRON
The test used potassium ferricyanide to detect the
presence of ferrous ion in the blood. The ferrous ion
reacts with the reagent to form Turnbull’s blue.

TESTS FOR FIBRIN Figure 8.20.

TEST FOR TRYPTOPHAN Turnbull's blue
The test used Hopkins-Cole reagent to detect the positive result for
presence of tryptophan, which is an amino acid potassium
monomer found in the structure of fibrin. The ferricyanide test.
positive result for this test is the formation of a
purple ring at the junction of two liquids.

TEST FOR BLOOD GASES

The test used Stoke’s reagent to detect the presence
Figure 8.17. Hopkin-Cole test positive of oxyhemoglobin and the reduced hemolglobin.
result. Stoke’s reagent with ammonium hydroxide forms a
powerful reducing agent that reacts with
oxyhemoglobin, changing the color of the blood
from intensely bright red into darker purple colored
solution.

Figure 8.21. Positive

TEST FOR SOLUBLE PROTEINS result for blood
The test used Millon’s reagent to detect the gases using Stoke's
presence of tyrosine, also an amino acid monomer reagent.
found in the structure of fibrin. The positive result is
the formation of red color in the solution.

URINE ANALYSIS

Figure 8.18. Millon's

test positive result.
 After the area has been thoroughly
cleansed, release some urine first before
collecting the sample needed.

Physical Tests
Color
 Assess the color of the urine while still in
A urinalysis is used to detect and manage a wide the urine cup.
range of disorders, such as urinary tract infections,  Most common colors of urine include
kidney diseases, and diabetes. Several compounds colorless, yellowish to amber, red or pink,
found in urine can be detected through physical and orange, blue or green, or dark brown.
chemical analysis.  Record observations.
Odor
PROCEDURE OF THE EXPERIMENT  Assess the odor of the urine.
Below is the procedure on how to perform urinalysis  Urine odor may be described as offensive,
using urine dipstick. Read and analyze the processes sweet, or unremarkable.
carefully.  Record observations.
Transparency
MATERIALS, APPARATUS, AND REAGENTS  Assess the transparency of the urine.
Test Samples Materials & Apparatus  Urine transparency may be described as
Urine Latex gloves clear or turbid (cloudy). Note if the urine is
Paper towels or tissue also frothy.
Rubbing alcohol  Record observations.
Urine cup
Urine dipstick
Biochemical Test
Dipstick test will be used for the biochemical test of
PROCEDURES
the urine sample. The chemical strips change color if
Preparing the Urine Sample: Precautions
certain substances are present or if their levels are
above normal. The test zone interacts with the urine,
All students who will provide the urine sample
producing results. Test zone is shown on Figure 8.22.
should avoid any intense athletic activity before the
test, as it may cause small amounts of blood to
appear in the urine. Many urinary constituents are
labile, and samples should be tested within one hour
of collection or refrigerated at 2-8°C for up to 24
hours for chemical analysis.

Collecting the Urine Sample

The urine sample should be collected in a urine cup
with tight-fitting lid. A randomly voided sample is
suitable for urine analysis, although the urine that is
Figure 8.22. Test zone in a urine dipstick.
first voided in the morning is preferable because it is
the most concentrated one. The best sample for
 Check first for the expiry date of the urine
analysis is collected using the midstream void (clean-
dipstick. If it is already beyond the expiry
catch) method. To collect a sample using the clean-
date, ask for a new batch of urine dipstick.
catch method:
 Take one test strip from the container.
 Females should use a clean cotton ball
Avoid touching the test zones as this may
moistened with lukewarm water (or
cause contamination, leading to faulty
antiseptic wipes) to cleanse the external
results.
genitalia before collection. To prevent
 Immerse the test strip in the urine sample,
contamination with menstrual blood,
making sure that all the test zones are
vaginal discharge, or germs from the
completely immersed.
external genitalia, release some urine first
 Once the test strip has been saturated with
before collecting the sample needed.
urine, remove the test strip from the urine
 Males should use a piece of clean cotton
sample, and place it on top of a paper
ball moistened with lukewarm water (or
towel. Tilt the strip horizontally on top of
antiseptic wipes) to cleanse the head of the
the paper towel and let the urine drip
penis and the urethral meatus (opening).
 gently from the test strip. Let the urine drip test strip. Any color changes that take place
off the side of the test strip, not down its after the initial two minutes should be
length. Never shake the dipstick or blot it disregarded, as the longer the urine remains
with another object. exposed, the more likely it is to produce
 Turn the strip sideways, as shown in Figure false-positive results.
8.23, before reading it to make sure that
the reactive chemicals in the test strip
won’t run from one square to another. Be
sure to keep the test squares facing up so
they will be plainly visible.

Figure 8.25. Read test squares in chronological order.

9. Interpret the results carefully. Different
Figure 8.23. Tilt the dipstick sideways. colors indicate the presence of different
6. Wait approximately 2 minutes for the substances. High amounts of proteins, for
results. It can take anywhere from 30 to 120 example, will turn the corresponding
seconds for the compounds in the urine to protein square (PRO) a cyan blue, while
react with the reagents on the test squares. elevated nitrite levels (NIT) are common
Read the instructions for the specific test with UTIs. Refer to the color chart
being performed to know the exact time frequently to get a better sense of the
needed. Once the reaction is underway, the significance of each value. Look at the pH,
squares will gradually change its color. Set a specific gravity, and glucose levels (GLU) of
timer to know exactly when the test is a urine sample, regardless of what is being
complete. screened for. The leukocyte and ketone
7. Compare the test squares to the color of ranges can point to potentially serious
the chart found in the packaging bottle of conditions like bacterial infections or even
the urine dipstick. diabetes.
10. Once the interpretation is done, discard the
test strip used into a clinical waste bin along
with gloves.
URINE
Urine, a typically sterile liquid by-product of the
body, is secreted by the kidneys through a process
called urination and excreted through the urethra.
Urine is often used as a diagnostic feature for many
disease conditions. These may be based on either
physical or chemical components, that may give
insight to processes within the body, often through
urinalysis, a common clinical analysis of urine.
Figure 8.24. Compare the color of the test strip with
the chart on the bottle.
Urine is an aqueous solution of greater than 95%
water. Other constituents include urea, chloride,
8. Read the test squares in chronological
sodium, potassium, creatinine and other dissolved
order, as shown in Figure 8.25. The squares
ions, and inorganic and organic compounds.
on a test strip are designed to react
sequentially. This makes trying to examine
the findings less chaotic. It will typically take
about half a minute before starting to
observe any changes. Check the value of the
first square (the one closest to the hand),
then move on to the next and proceed from
there until you have reviewed the entire
Figure 8.26. A urine sample.
Figure 8.27. Three types of urine analysis.
Urine sterile until it reaches the urethra. The
epithelial cells lining the urethra are colonized by A urinalysis involves checking the appearance,
facultative anaerobic gram (–) rods and cocci, concentration and content of urine. Abnormal
thereby making it unsterile once it is expelled out of urinalysis results may point to a disease or illness. A
the body. One of the waste products excreted routine urinalysis usually includes the following
through urine is urea, a processed form of ammonia tests: color, transparency, specific gravity, pH,
that is non-toxic to mammals. protein, glucose, ketones, blood, bilirubin, nitrite,
urobilinogen, and leukocyte esterase.
Urine is produced not only to eliminate many cellular
waste products, but also to control the amount of Routine urinalyses are performed for several
water in the body. In a way, urine volume regulation reasons:
is part of homeostasis, in that it directly regulates  General health screening to detect renal
blood volume, because greater amounts of urine will and metabolic diseases
reduce the volume of waters in blood.  Diagnosis of diseases or disorders of the
kidneys or urinary tract
URINALYSIS
 Monitoring of patients with diabetes
A urinalysis (UA), also known as routine and
microscopy (R&M), is an array of tests performed on
Routine urinalysis consists of three testing groups:
urine, and one of the most common methods of
physical characteristics, biochemical tests, and
medical diagnosis. Urinalysis means the analysis of
microscopic evaluation.
urine, and it is used to diagnose several diseases
such as urinary tract infections, kidney disease and
URINE SAMPLE COLLECTION
diabetes.
 24-hour collection – patient voids into
toilet, then all urine is collected for the next
The target parameters that are measured or
24 hours; as the body chemistry alters
quantified in urinalysis include many substances and
constantly, this is used to measure
cells, as well as other properties, such as specific
substances, such as steroids, white cells,
gravity. A part of a urinalysis can be performed by
electrolytes or determine urine osmolarity
using urine test strips, in which the test results can
 First-morning specimen – first specimen of
be read as the strip’s color changes. Another method
morning (or eight hours after recumbent
is light microscopy of urine samples.
position); best sample for pregnancy testing
 Fasting specimen – the second voided
When doctors order a urinalysis, they will request
specimen after a period of fasting
either a routine urinalysis or a routine and
 Mid-stream urine (MSU) – used to obtain
microscopy (R&M) urinalysis; the difference being
urine for bacterial culture; first and last part
that a routine urinalysis does not include microscopy
of urine stream is voided into the toilet to
or culture. R&M is used specifically for culturing
avoid contaminating the specimen with
bacteria found in urine, which can make it an
organisms presenting on the skin
important tool for diagnosing specific urinary tract
 Random specimen – for chemical or
infections.
microscopic examination, a randomly
collected specimen suitable for most
screening purposes
 Catheter specimen of urine – collected for
bacteriological examination if a patient’s
symptoms suggest the presence of a UTI;
the sampling technique used for collection
is important
URINE DIPSTICK caused by excessive amounts of prophobilin or
Urine dipstick comprises up to 10 chemical pads urobilin (a chemical produced in the intestines).
which react through change in color when immersed
in urine. The strips consist of a plastic ribbon of
about 5 mm wide and have pads impregnated with
reagent that react with the compounds in urine. The
test give a qualitative (+ or –) result, but some are
semi-quantitative.

The test is very simple to do. The test strip is

submerged in a urine sample. Excess urine is
removed by placing the dipstick in a paper towel,
tilted sideways and not vertically. It can take from 30 Figure 8.29. Urine color.
to 120 seconds for the compounds in the urine to
react with the reagents on the test squares. The Urine Transparency
squares on a test strip are designed to react Normal urine is transparent. Turbid (cloudy) urine
sequentially; this makes trying to examine the may be caused by either normal or abnormal
findings less chaotic. processes. Normal conditions giving rise to turbid
urine include precipitation of crystals, mucus, or
vaginal discharge. Abnormal causes of turbidity
include the presence of blood cells, yeast, and
Figure 8.28. Urine bacteria. It could be gauged subjectively and
dipstick. reported as clear, slightly cloudy, cloudy, opaque or
flocculent.

PHYSICAL TESTS
Physical characteristics that can be applied to urine
include color, turbidity (transparency), smell (odor),
pH (acidity – alkalinity) and density. Many of these
characteristics are notable and identifiable by vision
alone, but some require laboratory testing.
Figure 8.30. Urine transparency.
Abnormalities in any of these of physical
characteristics may indicate disease or metabolic Urine Odor
imbalances. These problems may seem superficial or The smell of urine may provide health information.
minor on their own, but can actually be the The urine of diabetics may have a sweet or fruity
symptoms for more serious diseases, such as odor due to the presence of ketones or glucose.
diabetes mellitus, or a damaged glomerulus. Generally, fresh urine has a mild smell, and aged
urine has a stronger odor similar to that of ammonia.
Urine Color
Normal urine is straw yellow to amber in color. Urine Density
Abnormal colors include bright yellow, brown, black The specific gravity of urine is a measure of the
(gray), red, and green. These pigments may result concentration of dissolved solutes (substances in a
from medications, dietary sources, or diseases. For solution), and it reflects the ability of the kidneys to
example, red urine may be caused by blood or concentrate the urine (conserve water). Specific
hemoglobin, beets, medications, and some gravity is usually measured by determining the
porphyria. Black-gray urine may result from melanin refractive index of a urine sample (refractometry) or
(melanoma) or homogentisic acid (alkaptonuria, a by chemical analysis. Specific gravity varies with fluid
result of a metabolic disorder). Bright yellow urine and solute intake. It will be increased (above 1.035)
may be caused by bilirubin (a bile pigment). Green in persons with diabetes mellitus and persons taking
urine may be caused by biliverdin or certain large amounts of medication. It will also be increased
medications. Orange urine may be caused by some after radiologic studies of the kidney owing to the
medications or excessive urobilinogen (chemical excretion of radiologic contrast dye. Consistently low
relatives of urobilinogen). Brown urine may be specific gravity (1.003 or less) is seen in persons with
diabetes insipidus. In renal failure, the specific
gravity remains equal to that of blood plasma  it is normal for urine to contain
(1.008–1.010) regardless of changes in the patient’s urobilinogen but not bilirubin
salt and water intake. Urine volume below 400 mL  indicative of RBC breakdown (not effectively
per day is considered oliguria (low urine production) removed by liver; liver damage)
and may occur in persons who are dehydrated and  indicative of problem with drainage of bile
those with some kidney diseases. A volume in excess into the gut (gallstones)
of 2 liters (slightly more than 2 quarts) per day is
considered polyuria (excessive urine production); it Ketone
is common in persons with diabetes mellitus and  formed during abnormal breakdown of fat
diabetes insipidus.  may result from prolonged vomiting, fasting
or starvation, on a diet, or with diarrhea
BIOCHEMICAL TESTS FOR URINE  also present in poorly controlled diabetes
BIOCHEMICAL TESTS patient
Biochemical testing of urine is performed using dry  it can make blood more acidic (diabetic
reagent strips, often called dipsticks. A urine dipstick ketoacidosis)
consists of a white plastic strip with absorbent
microfiber cellulose pads attached to it. Each pad Specific Gravity
contains the dried reagents needed for a specific  identifies hydration of an individual
test. The person performing the test dips the strip  a well-hydrated will have diluted urine (high
into the urine, lets it sit for a specified amount of fluid intake, diabetes insipidus, endocrine
time, and compares the color change to a standard disorders)
chart.  a dehydrated will have a concentrated urine
(low fluid intake)
 normal: 1.001–1.035
 when assessing, environmental factors such
as temperatures should be taken into
account

Blood
 normal: urine does not have blood
 abnormal: hematuria
 macroscopic: large volumes; dark colored
 microscopic: undetectable in naked eye;
reagent strip or microscope are needed
 enters urine due to damage in the filtration
barriers in kidneys (kidney damage)
Figure 8.31. Urine dipstick color chart.  also indicative of inflammatory lesions of
the urinary tract and kidney stones
Glucose  urine can also be contaminated with
 normal: urine does not contain glucose menstrual blood
 abnormal: glycosuria
 it occurs in pregnancy or patients taking pH
corticosteroids  measure of acidity or alkalinity (urine is
 indicative of diabetes mellitus slightly acidic)
 indicative of endocrine abnormality  normal: range of 5.0–8.0
 not diagnostic; further testing by blood test  too acidic urine may indicate kidney stones
(FBS)  too basic urine may indicate UTI caused by
Proteus, Klebsiella or Pseudomonas
Bilirubin & Urobilinogen  also affected by diet (high protein causes
 bilirubin is a product of RBC hydrolysis acidic urine; high intake of dairy products or
 it is transported in the blood to the liver vegetables causes alkaline urine)
where it is processed and excreted into the  results should be interpreted in conjunction
gut as a constituent of bile with an individual’s specific presentation
 in the gut, bacteria acts on bilirubin to
transform it into urobilinogen Protein
  normal: urine does not contain high level of abnormal metabolic process and are always clinically
protein significant. Normal crystals are formed from normal
 abnormal: proteinuria metabolic processes; however, they may lead to the
 protein is a large molecule to pass through formation of renal calculi, or kidney stones.
the glomerular filtration barrier
 can be caused by glomerular filtration
barrier damage or disease, kidney damage,
hypertension, diabetes mellitus
 specific investigations will be required to
detect the cause of proteinuria

Nitrite
 normal: urine does not contain nitrite
 presence are ad with presence of bacteria
that converts nitrate to nitrite
 may indicate UTI but clinical presentation of
symptom should be observed Figure 8.32. Urine sample under the microscope.
 absence of nitrites however does not
always rule out the presence of UTI
 approximately 50% of urine samples
containing bacteria, the nitrite test was
negative

Leukocytes
 usually associated with UTI but may indicate
a more severe renal problem
 when WBCs are present, patients are said to
have pyuria (pus in the urine)
 to establish, sample must be examined
under microscope and cultured
 where no bacterial cells are detected,
patients have sterile pyuria (which occurs in
tuberculosis and kidney inflammatory
disease)

MICROSCOPIC TESTS FOR URINE

MICROSCOPIC TEST
A urine sample may contain cells that originated in
the blood, the kidney, or the lower urinary tract.
Microscopic examination of urinary sediment can
provide valuable clues regarding many diseases and
disorders involving these systems.

The presence of bacteria or yeast and white blood

cells helps to distinguish between a urinary tract
infection and a contaminated urine sample. White
blood cells are not seen if the sample has been
contaminated. The presence of cellular casts (casts
containing RBCs, WBCs, or epithelial cells) identifies
the kidneys, rather than the lower urinary tract, as
the source of such cells. Cellular casts and renal
epithelial (kidney lining) cells are signs of kidney
disease.

The microscopic examination also identifies both

normal and abnormal crystals in the sediment.
Abnormal crystals are those formed as a result of an