EXPERIMENT 8: ISOLATION AND CHARACTERIZATION OF DNA FROM ONION

Nucleic Acid
- monomer: nucleotides
o an individual nucleotide consists of 3 parts
 1. nitrogenous base
 2. Sugar
 3. phosphoric acid residue
o all of which are covalently bonded together
o bases are bonded to the sugars, forming nucleosides
- one of the essential biochemical molecules present in an
organism
- functions for encoding, transmitting, and expressing the
genetic information
- principal types are DNA and RNA

Nitrogenous Base
- Pyrimidines: single-ring aromatic compounds
- Purines: double-ring aromatic compounds

DNA
- It is an essential biochemical molecule of an organism
that contains genetic information
- It is responsible for the existence of our inherited
physical traits.
- is made up of molecules called nucleotides
- The Structure of DNA
- Double helical structure
- Proposed in 1953 by James Watson and Francis Crick
- It was based primarily on model building and X-Ray
diffraction patterns

Chargaff’s Rule
- A=T
- G=C
- The amount of Adenine was always the same
amount with Thymine, and Guanine always has the same amount as
Cytosine.
- The reason A goes with T, while C goes naturally with G, is because of
their bases connect through hydrogen bonding.
- The sugar-phosphate backbone is the outer part of the helix.
- And as you can see the chains run in antiparallel directions.

The Double Helix

- Composed of complementary strands
- It is possible that A will pair with C and T will pair with G, but the
hydrogen pairing will be incorrect. Purine-Purine and Pyrimidine-
Pyrimidine is also possible but the dimension will not be correct.
- The atoms that make up the polynucleotide chain leave empty spaces known as grooves.
Onion (Allium cepa)
Why onion?
- It has low starch content and longer strands which allow us to see the DNA more clearly and spool out more
easily
- has more DNA than most species

Why white over violet onion?

- Because it has longer strands of DNA
Objectives
1. To isolate DNA from Allium cepa (onion)
2. To determine the concentration and purity of the isolated
DNA,
3. To cleave the DNA via acid hydrolysis and,
4. To analyze the components of DNA hydrolyzate

METHODOLOGY
A. Isolation of DNA from onion
Preliminaries:
1. Heat the tip of a dropper in a Bunsen flame until you can bend
the end back up towards the top forming a hook. This will be
used to spool and hook the DNA.
2. Cool 50mL of 95% ethanol in an ice bath
B. Determination of DNA Concentration and Purity

C. Acid Hydrolysis of DNA

D. Chemical Characterization of DNA
DISCUSSION
Preliminaries
Why was it necessary to remove the first few layers and mince the onion?
- Cell lysis or cellular disruption
- To extract biological molecules including organelles, proteins, DNA, RNA and lipids from inside the cell

- *Chopping the onion allows its tissues to be broken up so that homogenizing solution can take effect and attack
the cell walls and membranes.

A. ISOLATION OF DNA FROM ONION

1. Homogenization
- Break up the onion tissues to separate and open cells
- Denaturation of DNAse enzymes
- Breaking down of cell wall, cell membrane, and nuclear membrane allowing the release of DNA to a medium in
which it is soluble and protected from degradation

- *In this step, we will break up the onion tissues to separate and open cells.
- Cell walls, and cell and nuclear membranes get fragmented, and molecules contained within the cells (including
DNA) are liberated into the medium.
- We will briefly heat the tissue before homogenization, in order to inactivate DNAses (enzymes that degrade
DNA) that are present in our mixture.
- Once we degrade DNAses, we will maintain the homogenate at a cold temperature, in order to keep enzymes
deactivated and the DNA cold for the precipitation step.
- After we homogenize the onion tissue in the blender, we will strain it through cheesecloth to remove the bigger
debris, such as cell walls, membranes, etc.

2. Deproteinization
- hydrolysis of protein using Papain powder
- purify the homogenate by removing proteins that are associated with cells and DNA molecules

- *In this step, we will purify the homogenate by removing proteins and lipids that are associated with cells and
DNA molecules.
- We will take advantage of the differential solubility of DNA, proteins, lipids and other macromolecules, to
separate them using different solvents.
- DNA is soluble in water, but other macromolecules are more soluble in organic solvents.
- If we add chloroform (one such organic solvent) to our homogenate (which contains water), two layers will form
like in an oil/vinegar dressing.
- Those molecules that are soluble in water will remain in the top water layer, and those molecules that are
chloroform-soluble will drop to the bottom chloroform layer.

3. Precipitation
- Force DNA to come out of solution, or precipitate, by using cold alcohol
- Adding ethanol alcohol which causes every component in the filtrate to stay in solution except DNA

- In this step we will force DNA to come out of solution, or precipitate, by using very cold alcohol.
- When precipitation is completed, the liquid within the flask will become cloudy.
- This cloudiness is really the long nucleotide polymer (DNA).
- Once we are at this step, we will be able to spool the long DNA strands onto a very cold glass rod and out of the
beaker.
Why heat in a water bath until the solution reaches 60
degrees Celsius?
- Heating at 60C softens the phospholipid in the cell
membrane and denatures DNAse
- Why should the temperature be kept at 60
degrees Celsius?
o To prevent the denaturation of DNA that
involves the breaking of hydrogen bonds
between the bases in the duplex
Papain
- Proteolytic enzyme
- Degrades DNAses, RNAses, and proteins
- Denatures the proteins clinging to the DNA making
the molecule flexible and easy to spool
Why use 95% ethanol?
- When the DNA is liberated from the nucleus and
the cell, alcohol must be added to recover it. 95%
alcohol is less dense than water and so floats on
the surface. The lipids and proteins will fall to the
bottom of the test tube while the DNA, which is
less dense than the proteins and lipids, will rise
into the alcohol layer.
Why transfer the flask immediately into an ice bath?
- Slows down the DNA breakdown
- At room temperature, DNA begins to denature by
the action of DNAse (present in cell extracts)
Why is the onion tissue mixed in a blender?
- To allow the release of DNA with homogenization
media which breaks down the cell wall, cell
membrane, and nuclear membrane
- Why 45 seconds only?
o Exceeding would expose the DNA to…
forces that rapidly break the DNA into
shorter and shorter length
Homogenizing solution
- Separate the DNA from chromosomal protein
through its chemical components which will cause
the proteins to precipitate out of solution
- • *to further dissociate the complexes and
Filtering of the homogenate through a cheesecloth
- To remove bigger debris, such as cell walls,
membranes, or to remove any solid material,
resulting to a clear cell homogenate
- More efficient compared to filtrate
Why use an ice-col 95% ethanol?
- This is added to cause the DNA to precipitate out
of the solution
- 95% ethanol is recommended since DNA is not
soluble in alcohol and the colder the alcohol, the
less soluble it is
- The DNA precipitated out of this solution using salt
and ethanol
o Salt neutralizes the charges on the
phosphate groups in the DNA backbone
o The alcohol, having a lower dielectric
constant than water, then allows the
sodium ions from the salt to interact with
the negatively charged phosphate groups
closely enough to neutralize them and let
the DNA fall out of the solution
- Since DNA strands are negative, ethanol being an
alcohol, which is non-charged, acts to repel the
negative DNA
Purpose of the following in the isolation of DNA
• SDS (sodium dodecyl sulfate)
• Detergent used to break down nuclear
membrane
• Anionic detergent which emulsify lipid and
protein components of the cell by
disrupting the noncovalent interactions
that hold the cell membrane together
• Detergent form complexes with these
lipids and proteins causing them to
precipitate out of the solution
• Chelating detergent
• Sodium citrate
• Provide sodium
ions that
neutralize the
negative charge of the DNA backbone
(phosphates)
denature the
enzymes
• EDTA
(Ethylenediaminetetraacetic
acid)
• Inactivates DNAse (breaks down DNA)
B. DETERMINATION OF DNA CONCENTRATION AND
PURITY
Method used: Spectrophotomeric quantification of
DNA
Principle: Ultraviolet absorption
 Major absorption band for purified DNA
peaks at 260nm
 Protein material has a peak absorption
at 280nm
 Typical A260/A280 is 1.8
UV/Vis Spectroscopy
- determined the purity of the isolated DNA
- absorbance at 260nm is due to the DNA species
- absorbance at 280nm is due to the protein
species
C. ACID HYDROLYSIS OF DNA
• Hydrolysis of glycosidic bonds to the purine
bases at pH4
• Protonation of purine bases
• Isomerization of depurinated sugar into its open
chain form
Reagents:
- 1.0M HCL
o Causes the dissociation of DNA into its
components
o Causes depurination
- 1.0M NaOH
o Neutralizes the solution
o Needed so that it wouldn’t affect the
tests
- Absorbance ratio (A260/A280)
- gives the relative measurement of DNA and
protein content in isolated DNA
- A260/A280 = 1.8-1.9 pure DNA, free of protein
contamination
- A260/A280 < 1.8-1.9 protein contamination
- A260/A280 >1.8-1.9 presence of RNA
-
*Factors that affect UV absorption
 Presence of aromatic bases adenine,
guanine, cytosine, and thymine
 Denaturating agents (Saline Sodium
Citrate)
Why not used alkaline hydrolysis?
- Pentose of DNA doesn’t have an –OH group
at the 2nd carbon
- No formation of monophosphate
intermediate
- DNA is stable in alkaline hydrolysis
Why is the DNA-HCl mixture heated at 100C?
- To be able to destroy the hydrogen,
phosphodiester, and glycosidic bonds
- Results to the dissociation of DNA into its
components:
o Phosphate group
o Purine and pyrimidine
o Deoxyribose
Result of Hydrolysis
- Breaking of bonds associated with the DNA
molecule
- Hydrogen bonds
 Heating to temperatures above
80C
- Phosphodiester bonds
 Heating to temperatures above
90C
 Acids, with pH less than 2
- Glycosidic bonds
 Acids, with pH less than 2
D. CHEMICAL CHARACTERIZATION OF DNA
Discussion: D.1 Test for Deoxyribose
• “Dische Test”
• Standard: Deoxyribose standard solution
• Reagents: Diphenylamine reagent (contains conc. H2SO4)
• Positive Result: blue colored solution
• Positive in: Deoxyribose standard solution and DNA hydrolyzate
Principle:
 1. Dehydration of deoxyribose by H2SO4
 Formation of 5-hydroxy-levulinaldehyde
 2. Complexation with diphenylamine
 Gives a blue colored complex
 The intensity of the blue color is proportional to the concentration of the DNA.
- *The reaction between the dische reagent and 2-deoxypentose results in the development of a blue color.
- The reaction depends on the conversion of the pentose to hydroxylaevunilic aldehyde which then reacts with
diphenylamine to give a blue colored complex.
- The intensity of the blue color is proportional to the concentration of the DNA. The sample produced a positive
result.

Discussion: D.2 Test for Phosphate

• Standard: Phosphate solution
• Reagents:
o conc. H2SO4
o conc. HNO3
o 2.5% Ammonium molybdate solution
• Positive Result: yellow precipitate (ammonium phosphomolybdate)
• Positive in: phosphate solution and DNA hydrolyzate
Principle:
 Hydrolysis of pyrophosphate to phosphate
 Precipitation of phosphate
 Forms phosphoammonium molybdate which gives a positive result of yellow ppt

Discussion: D.3 Test for Purines

• “Murexide Test”
• Standard: solid guanine
• Reagents:
o conc. HNO3
o 10% KOH
• Positive Result: red purple residue
• Positive in: solid guanine
• Discussion: D.3 Test for Purines
Principle:
 1. Oxidation of purine by conc. HNO3
 Forming dialuric acid and alloxan
 2. Condensation reaction of alloxan to form alloxanthin
 3. Neutralization, forming red purple murexide (potassium salt of purpurate)

- * A color test that is positive for all purines capable of oxidation to an alloxan derivative
- DNA reacted with nitric acid since purines is known to be readily soluble in diluted acid. Nitric acid oxidized it
leaving a yellow precipitate. The sample produced a yellow precipitate which indicates a positive result.
Discussion: D. 4 Test for Pyrimidines
• “Wheeler-Johnson”
• Standard: Cytosine solution
• Reagents:
• Bromine water
• Ba(OH) 2
• Positive Result: purple precipitate
• Positive in: Cytosine solution

Principle:
 1. Bromination of pyrimidine to form dialuric acid
 2. Neutralization of dialuric acid by Ba(OH)2 Bromination = converts pyrimidines into
dibromohydrouracil
 to form Barium salt of Dialuric Acid

- *Bromine water reacted with the sample to form 5- bromo-6- hydroxyhydroxo derivative which provides a green
coloration.
- Upon addition of Ba(OH)2 will give a result of purple precipitate.
- The sample produced a white cloudy solution which is a negative result

TEST REAGENTS TEST FOR PRINCIPLE (+) RESULT (+) IN

Dische Diphenylamine Deoxyribose 1. dehydration of deoxyribose blue colored Deoxyribose
Reaction reagent/conc. by H2SO4 solution standard solution
H2SO4) 2. Complexation with and DNA
diphenylamine hydrolysate
Test for - conc. H2SO4 Phosphate Precipitation of phosphate Yellow ppt phosphate
Phosphate - conc. HNO solution and DNA
3
hydrolyzate
- 2.5%
Ammonium
molybdate
solution
Murexide - conc. HNO3 Purines 1. Oxidation of purine by Red purple Solid guanine
Test -10% KOH (Adenine conc. HNO3 residue
and 2. Condensation reaction of
Guanine) alloxan
3. Neutralization, forming red
purple murexide
Wheeler- - Bromine Pyramidines 1. Bromination of pyrimidine Purple ppt Cytosine solution
Johnson water (Cytosine to form dialuric acid
- Ba(OH) 2 and 2. Neutralization of dialuric
Thymine) acid by Ba(OH) 2 to form
Barium salt of Dialuric Acid
EXPERIMENT 9: URINALYSIS
Urine
- By product secreted by the kidneys
- Aqueous solution of metabolic wastes such as:
urea, dissolved salts, organic compounds etc.
- Reflects the work of the kidneys to maintain
homeostasis
- Transported by the ureter to the urinary bladder
and voided through urethra
- Urine is a typically sterile liquid by-product of the
body secreted by the kidneys through a process
called urination and excreted through the urethra.
- Urine is an aqueous solution of greater than 95%
water. Other constituents include urea, chloride,
sodium, potassium, creatinine and other dissolved
ions, and inorganic and organic compounds.
- Abnormal constituents found in urine are an
indication of disease.
- The presence of red blood cells in urine is referred
to as haematuria.
- Red blood cells in urine can indicate inflammation,
problems with the kidneys or urinary tract, or even
cancer.
- The presence of haemoglobin in urine indicates
hemolysis of red blood cells and passage through
the kidney tubules.
- The presence of white blood cells in urine can
indicate kidney or urinary tract infection.
- The presence of proteins, which are normally too
large to pass through the tubules, can be an
indication of damage to the tubules.

- Composition of Urine
o 0.05% Ammonia o 0.1% Magnesium o 0.1% Creatinine
o 0.18% Sulfate o 0.015% Calcium o 0.03% Uric Acid
o 0.12% Phosphate o 0.6% Potassium o 2% Urea
o 0.6% Chloride o 0.1% Sodium o 95% Water

PHYSICAL EXAMINATION OF URINE

1. Color
- Color of urine is attributed to the presence of three coloring pigments, namely, urochrone, urobilin, and
uroerythrin.
o Color comes from the presence of urobilin
o Urobilin
 final waste product resulting from the breakdown of heme from hemoglobin during the
destruction of blood cells
- Rough indication of the state of hydration of an individual
- The darker the color of urine, the more concentrated it will be. Which signals that one has to drink plenty of
water for he/she may already be dehydrated.
- Normal: clear yellow to yellow orange

2. Odor
- Ordinarily, odor has little diagnostic significance and is not included in the routine laboratory result
- Reflection of what has been consumed or specific diseases

3. Turbidity
- Refers to the degree of cloudiness in a urine
- Otherwise known as clarity or transparency
- Normal: clear and transparent
- Turbid/cloudy urine indicates:
- Symptom of a bacterial infection
- Caused by crystallization of salt such as calcium phosphate
4. Volume Polyuria Excessive urine excertion; seen in increased
- Indicates balance between fluid ingestion and water water ingestion, diabetes; >2.5L /a day
lost from lungs, sweat, and intestines
Oliguria Scanty urine excertion; seen in dehydration,
- 1-2 L/a day is the normal adult volume
renal failure; <400 mL/day

5. pH anuria Absence of urine output; seen in renal shut

- Reflects ability of kidney to maintain normal hydrogen down, <100 mL/ day
ion concentration
- In a pH balanced body. urine is slightly acid in the morning, (pH = 6.5 - 7.0) generally becoming more alkaline (pH
= 7.5 - 8.0) by evening in healthy people primarily because no food or beverages are consumed while sleeping.
- Tested using: pH paper, litmus paper

CHEMICAL ANALYSIS OF URINE

The chemical analysis of urine is undertaken to evaluate the levels of the following component:
- Protein
o The presence of increased amounts of protein in the urine can be an important indicator of renal
disease.
o When urine protein is elevated, a person has a condition called proteinuria; this can be an early sign
of kidney disease.
- Glucose
o The presence of significant amounts of glucose in the urine is called glucosuria.
o Quantity of glucose that appears in the urine is dependent upon the blood glucose
level, the rate of glomerular filtration, and the degree of tubular reabsorption.
o When the blood glucose exceeds the renal threshold, the tubules cannot reabsorb
all of the filtered glucose, and so glycosuria occurs.
o Usually, glucose will not be present in the urine until the blood level exceeds 160–180 mg/dL, which is
the normal renal threshold for glucose
- Ketones
o Ketones, or ketone bodies are formed during the catabolism of fatty acids.
o An abnormal result means you have ketones in your urine.
 Small: <20 mg/dL
 Moderate: 30 to 40 mg/dL
 Large: >80 mg/dL
- Occult Blood
• term “occult” means “hidden,”
• procedures actually detect the free hemoglobin from lysed red blood cells (RBCs)
• chemical methods used in the routine urinalysis for detecting blood (hematuria) will also detect free
hemoglobin (hemoglobinuria) and myoglobin (myoglobinuria)
• urine is normally free of all of these substances; therefore, a positive test for occult blood should be
followed by determination of the exact cause and origin of this abnormal finding
- Bilirubin C33H36N4O6
• formed from the breakdown of hemoglobin in the
reticuloendothelial system
• A yellowish pigment found in bile, a fluid produced by the liver
• Normally not found in urine
• Increased levels of bilirubin in the urine may be due to:
hepatitis, liver disease, tumors of the liver or gallbladder etc.
 - Urobilinogen C33H44N4O6
• normally present in urine in low concentrations
• A small amount of this urobilinogen is also excreted by the kidneys into
the urine, with a normal level of about 1–4 mg/24 h
• formed in the intestine from bilirubin, and a portion of it is absorbed
back into the bloodstream
• Positive test results help detect liver diseases such as hepatitis and conditions associated with increased
RBC destruction
Microscopic Examination
- One or two drops of urine sediments from a centrifuged urine is placed on a glass slide
- Both low power and high power objectives of the microscopes are used

Objectives
- The experiment aims to subject the urine samples acquired to several tests and to qualitatively examine the
presence of some normal organic constituents and pathological organic constituents.

METHODOLOGY
A. Initial Examination of the Urine Sample

B. Qualitative Examination for Normal Organic Constituents

C. Qualitative Examination of Pathologic Organic Constituents
DISCUSSION
B. QUALITATIVE EXAMINATION
1. Test for Urea
- Urea is a product of the conversion of ammonia and carbon dioxide.
- Ammonia is a dangerous substance in the body that is converted by the liver by using some enzymes and
molecules. Presence of large amount of urea is also unwanted, but much less harmful than ammonia.
- Elevated urea levels can occur with dietary changes, diseases which impair kidney function, liver diseases,
congestive heart failure, diabetes and infections
- Urea Cycle
o The presence of urea in the urine sample indicates
that no abnormality is present. The conversion of
urea from ammonia and carbon dioxide is called
the urea cycle or ornithine cycle. This cycle helps
the harmful ammonia to convert into urea for its
decomposition.

- test for substances that are readily/always present in

every urine sample
- Reagents: 70% NaOH and Bromine water
- Principle: Decomposition of (NH2)2 CO by NaBrO to N2 gas
- Positive result: evolution of N2 gas
 STEP 1:
- Takes place in the mitochondria
- 1st amino group formed in the matrix:
Ammonia from amide nitrogen of glutamate
- Carbamoyl phosphate enters the cycle
as carbamoyl group donor

STEP 2:
- Carbamoyl phosphate donates it carbamoyl group
to ornithine to form citrulline
- Citrulline is passed to cytosol

STEP 3:
- 2nd amino group enters: Aspartate
- Condensation reaction: amino group (Asp) +
carbonyl group (Citrulline)  argininosuccinate

STEP 4:
- The only reversible step in the cycle
- Fumarate enters mitochondria to participate in Kreb’s cycle

2. Test for Uric Acid

- Uric acid is an organic compound of carbon, nitrogen, oxygen and hydrogen with the formula C5H4N4O3.
Xanthine oxidase oxidizes oxypurines such as xanthine and hypoxanthine to uric acid.
- Uric acid is the final breakdown product of purine catabolism.
- A presence of this in the urine sample indicates that no abnormality is present.
- Diseases related to elevated uric acid levels are kidney stones, Lesch – Nyhan syndrome, CVDs and diabetes. Uric
acid reacts with phosphotungstic acid to produce allantoin
and tungsten blue.
- Test for Uric Acid

- Reagents: 20% Na2CO3 and Phosphotungstic acid reagent

- Principle: Oxidation and reduction of uric acid by
phosphotungstic acid
- Positive result: Formation of a blue colored solution
3. Indican Test
- Indican is an indole produced by bacterial action on an amino acid, tryptophan, in the intestine.
- In normal urine, the amount of indican excreted is small. It is increased with high protein diets or inefficient
protein digestion. If not digested properly, or if the wrong types of proteins are ingested, bowel putrefaction can
occur. Problems with protein digestion can be caused by overgrowth of anaerobic bacteria, intestinal
obstruction, stomach cancer, low stomach acid, parasitic infections, malabsorptive syndromes, fungal infections,
lack of digestive enzymes, or liver problems.
- Following absorption, indole is converted to 3-
hydroxy indole in the liver. Detection of indican in
the urine depends upon its decomposition and
subsequent oxidation of indoxyl to indigo blue
and its absorption into a chloroform layer.

- Reagents: Obermayer’s reagent and chloroform

(CHCl3)
- Principle: Oxidation of indoxyl by ferric ions
- Positive result: Formation of blue color in the lower layer

4. Test for Creatinine

- Creatine is formed when food is changed into energy through a process called metabolism. Creatine is broken
down into another substance called creatinine by the addition of strong acid or by alkali or by using enzyme,
creatine hydroxylase.
- If the kidneys are damaged and cannot work normally, the amount of creatinine in the urine goes down while its
level in the blood goes up.
- Alkaline picrate solution is composed of saturated picric acid and 10% NaOH. Most methods used for creatinine
determination are based upon the Jaffe reaction. The Jaffe reaction uses saturated picric acid which oxidizes
creatinine in alkali forming creatinine picrate, in which creatinine forms a characteristic orange color when
treated with alkaline picrate.

- Reagents: Alkaline picrate solution

- Principle: Creatinine reacts with picrate solution
- Positive result: Formation of orange colored solution

C. QUALITATIVE EXAMINATION FOR PATHOLOGIC ORGANIC CONSTITUENTS

1. Gunning’s Test
- The Gunning’s Test is a test for ketone bodies.
- The reagents used are the ammonium hydroxide and the Lugol’s solution which is composed of potassium
iodide and iodine.
- The ammonium hydroxide is used to basify the urine sample. The potassium iodide reacts with iodine to form
iodoform crystals (positive result).
- If the urine sample is negative, it indicates that there is enough insulin in the body

2. Benedict’s Test
- Benedict’s Test is a test for glucose.
- The reagents used are the Benedict’s reagent,which is composed of sodium citrate, sodium carbonate and
copper sulfate.
- The principle behind this is that there is a reduction of copper sulfate to cuprous oxide when exposed to alkali
and heat. The positive result for this is the formation of yellow to red precipitate, which indicates a possibility of
having diabetes.
3. Exton’s Test
- Exton’s Test is a test for the presence of Albumin, which is a protein.
- The Exton’s reagent is composed of anhydrous sodium sulfate, which is a drying reagent and sulfosalicylic acid.
The sulfosalicylic acid is a precipitating reagent that can precipitate the Albumin to form a white precipitate with
cloudiness.
- When positive in this test, the kidney has a serious abnormality because proteins do not usually appear in urine.

4. Smith’s Test
- Bile pigments are being tested in the Smith’s Test.
- Iodine alcoholic tincture, which is composed of iodine crystals and sodium iodide in ethanol reacts with the bile
pigments that bring emerald green colored interphase. This may indicate an abnormality in the liver.
- Addition of a solution of alcoholic iodine to urine produces a green color. The green color indicates the presence
of bile.

5. Test for Occult Blood

- The Test for Occult Blood is for the presence of red blood cells in the urine sample.
- 95%ethanol with guaiac powder, hydrogen peroxide and glacial acetic acid are the reagents used.
- The hemoglobin can decompose the hydrogen peroxide and this results to the oxidation of the guaiac powder
that produces blue interphase.
- The addition of hydrogen peroxide to 5mL 95% ethanol and guaiac powder oxidizes the guaiac causing a color
change. A blue ring indicates a positive result.[8]
- When positive in this test, there is a problem in the kidney or in other parts of the urinary tract.