Biochem Oral Presentation

Qualitative Examination of Urine
for Normal Organic Constituents

and Pathologic Organic
Constituents using various tests
Solas, Karsten Sean

Tuazon, Maria Christine
Yu, Lauren, Therese
Urine
Produced by the kidney
Composed of various organic and
inorganic substances which are normally
waste products from metabolic processes
Pale yellow to amber color
Used in testing for physiological diseases
95% of urine is water, 5% is solutes
Urine (Composition)
0.05% Ammonia
0.18% Sulphate
0.12% Phosphate
0.6% Chloride
0.01% Magnesium
0.015% Calcium
0.6% Potassium
0.1% Sodium
0.1% Creatinine
0.03% Uric acid
2% Urea
95% Water
Urinalysis
Set of tests used in urine
A part of routine diagnostic and screening
evaluations performed to provide a general
overview of a persons health
Detect substances and cellular materials
Urinalysis (Importance)
To diagnose and monitor renal or urinary tract
diseases.
To detect metabolic or systemic diseases not
directly related to the kidneys.
Objectives
To make an initial examination of the urine
sample
To qualitatively examine the presence of
some normal organic constituents of the
urine sample
To examine for pathological organic
constituents of the urine sample.
Methodology
A. Initial Examination of Urine
Urine Sample
- Note the color, turbidity, acidity

and the time when the urine was
collected.
Initial
Observations
B. Qualitative Examination for the Normal Organic Constituents
B.1 Test for Urea
1 ml urine sample
- Add 0.5 ml of 70% NaOH.

- Add 4 drops of bromine water.
- Record and observe the result.
Formation of
bubbles
B.2 Test for Uric Acid
1 ml urine sample
- Add 5 ml of 20% to the sample. Mix

well.
- Add 5 drops of phosphotungstic acid
reagent. Mix well.
- Record and observe the result.
Blue Solution
B.3 Indican Test
5 ml urine sample
- Add 5 ml of Obermayers reagent to the

sample. Mix well.
- Add 3 ml chloroform.
- Shake and allow the chloroform layer to
settle.
- Observe the blue color in the lower or
chloroform layer.
Blue lower
(chloroform) layer
B.4 Test for Creatinine
2 ml urine sample
- Add 1ml of alkaline picrate solution to the

sample.
- Note the formation of an orange-colored
solution
Orange Solution
C. Qualitative Examination for Pathologic Organic Constituents
C.1 Gunnings Test (For Ketone Bodies)
5 ml urine sample /
Positive Control: 5 ml urine sample + 2 ml acetone
- Basify the 5 ml urine sample with 5 drops

concentrated ammonium hydroxide. Use
red litmus paper.
- Add Lugols solution to the basic urine
enough to produce a black cloud which
does not appear immediately. Let stand
for 5 minutes.
- Note the formation of iodoform crystals.
- Perform similarly on a positive control.
Compare the results obtained.
Iodoform Crystals
C.2 Benedicts Test (for Glucose)
1 ml urine sample /
Positive Control: 1 ml urine sample + 1 ml 1% glucose solution
- Add 5 ml Benedicts reagent to 8-10 drops of

urine sample in a test tube. Mix well.
- Heat in boiling water bath for 2 3 minutes.
Let stand and allow to cool.
- Note the formation of a precipitate.
Brick red
precipitate
C.3 Extons Test (for Albumin)
3 ml urine sample /
Positive Control: 3 ml urine sample + a pinch of albumin
- Mix 3 ml of the sample and 3 ml Extons

reagent in a test tube.
- Warm the solution if cloudiness appears.
- If cloudiness persists or increase upon
heating, albumin is present.
Cloudy Solution
C.4 Smiths Test (for Bile pigments)
5 ml urine sample /
Positive Control: 5 ml urine sample + 3-5 drop of pigs bile pigment
- Place 5 ml of urine sample in a test tube.

- Incline the test tube and overlay with 3 ml of
tincture of alcoholic iodine mixture.
- Observe an emerald green at the point of
contact.
Emerald green at
the point of contact
C.5 Test for Occult Blood
3 ml urine sample /
Positive Control: 3 ml acidified (acetic acid) urine + 3 drops of pigs blood
- Add 5 ml of 95% ethanol to a half spatula

guaiac powder in a test tube. Mix well.
- Add 5 ml of hydrogen peroxide to the
mixture.
- Add the 5 ml of this solution to 3 ml of
acidified urine.
- A blue ring indicates the presence of blood.
Formation of Blue
ring
RESULTS AND DISCUSSION
Group # Time Color Turbidity pH
collected
1 6:30am Yellow Clear 6

2 7:00am Light Clear 6
yellow
yellow
4 6:30am Yellow Turbid 5
5
8 6:03am Dark Clear 6
yellow
9 5:35am Light Turbid 7
Yellow
Sample
A.1 Time of Collection
First morning specimen : is the specimen of choice since the
urine is generally more concentrated and abnormal resuts may
be more obvious during examination
Randomly collected specimen: not regarded as specimens of
choice because of the possibility of dilution of the specimen
after patient has consumed fluids prior collection
Timed collection: required for quantitative measurement of
certain analytes
Midstream clean catch collection: decreases the incidence of
microbial and cellular contamination.
Sample
A.2 Color
The normal urine is
clear and pale yellow
Pigment urobilin causes
the yellow color
An unusual urine color
can be a sign of disease
Sample
A.3 Turbidity
Freshly voided urine is clear and transparent
Non pathological: Crystals, deposits,epithelial
cells, spermatozoa etc.
Pathologic: Bacteria, blood cells, pus etc.
Sample
A.4 pH
Normal urine pH ranges from 4.5 to 8.0
Urine is slightly acidic in the moring (6.5 - 7) while
more alkaline in the evening (7.5-8)
Either too high or too low can indicate the likelihood
of forming kidney stones
Acidic: Acidosis, dehydration, Diabetic
ketoacidosis, starvation
Basic: Kidney failure, Pyloric obstruction, Urinary
Tract Infection, Respiratory alkalosis
Group # Time Color Turbidity pH
collected
1 6:30am Yellow Clear 6

yellow
yellow
4 6:30am Yellow Turbid 5
5 - - - -
8 6:03am Dark Clear 6
yellow
9 5:35am Light Turbid 7
Yellow
B.1 Test for Urea

Group # Urine sample
1 Bubble formation, N2 gas
2 Clear yellow solution with bubbles
3 Bubble formation, clear yellow solution
4 Bubble formation, yellow clear solution
5
8 Bubble formation, clear colorless solution
9 Bubble formation
B. Qualitative Examination for
Normal Organic Constituents
Test for Urea

Major component in urine (2%)
Hepatic enzymes convert ammonia to urea
Produced in the livers excreted through the kidneys
in urine.
The test can help determine how well the kidneys are functioning,
and if your intake of protein is too high or low.
Reagents: 70%NaOH and Bromine Water
Positive Results: Formation of gas
Principle: When urea is treated with sodium
hypobromite, it decomposes to give nitrogen.
B.1 Test for Urea

1 Bubble formation, N2 gas
2 Clear yellow solution with bubbles
3 Bubble formation, clear yellow solution
4 Bubble formation, yellow clear solution
5
8 Bubble formation, clear colorless solution
9 Bubble formation

1 Blue solution at the top ***
2 Clear light blue solution
3 Light blue solution
5
8 Clear colorless solution
9 Blue solution ***

Test for Uric acid

C5H4N4O3
End product of catabolism of purine bases.
Formed from nucleic acids.
Excreted only in small amounts in urine
Hyperuricemia ---- Gout
Reagents: Phosphotungstic acid reagent
Positive Results: Blue solution
Principle: Uric acid is a reducing agent in alkaline
medium. It reduces phosphotungstic acid to tungsten blue

1 Blue solution at the top ***
3 Light blue solution
5
8 Clear colorless solution
9 Blue solution ***

B.3 Indican Test

1 Clear chloroform layer
2 Clear orange solution, orange precipitate
3 Blue lower layer, bubbles at interphase, light brown
upper layer
4 Clear yellow solution, blue lower layer
5
8 Light blue layer, clear orange upper layer
9 Upper orange solution, bubbles formation on interphase,

colorless solution lower layer
Indican Test
Indole produced by bacterial action on tryptophan
and amino acids in the intestine.
Elevated levels of indole indicate intestinal toxemia and overgrowth
of anaerobic bacteria in the body.
Detection depends on its decomposition and oxidation of indoxyl to
indigo blue
Increases with inefficient protein digestion or high protein intake.
Reagents: Obermayers reagent, chloroform
Positive Results: Blue chloroform layer
Principle: Oxidation of indoxyl to blue indigo and its
absorption into the chloroform layer
B.3 Indican Test

1 Clear chloroform layer
2 Clear orange solution, orange precipitate
3 Blue lower layer, bubbles at interphase, light brown
upper layer
4 Clear yellow solution, blue lower layer
5
8 Light blue layer, clear orange upper layer
9 Upper orange solution, bubbles formation on interphase,

colorless solution lower layer

1 Clear orange solution
2 Clear brown solution with clear light blue
lower layer and green interphase
3 Clear Orange solution
4 Orange solution with precipitate
5
9 Orange solution on upper layer***

Test for Creatinine
Also known as Jaffes Reaction
A constant constituent of urine
Waste product in the blood that passes through kidneys and
eliminated in urine.
By-product of muscle function. The more muscle, the more
creatinine produced.
Creatine phosphate
High levels indicate kidney disease
Reagents: Alkaline picrate solution (5:1, saturated picnic
acid- 10% NaOH)
Positive Results: Red-orange colored solution
Principle: Creatinine reacts with picric acid in the
alkaline medium to form a red to orange colored complex
of creatinine picrate.

2 Clear brown solution with clear light blue
lower layer and green interphase
3 Clear Orange solution
4 Orange solution with precipitate
5
9 Orange solution on upper layer***

Group Positive Control Urine Sample
#
1 Formation of yellow Iodoform Red-brown clear solution
crystals with precipitate
2 light yellow solution with white Yellow solution with
precipitate whitish precipitate
3 Light yellow solution with Golden solution with
precipitate precipitate
4 Yellow clear solution with Turbid solution with black
yellow precipitate precipitate
5
8 Colorless solution, with crystals Orange clear solution with
at the bottom white crystals at the
bottom
9 Iodoform crystals Red-orange solution
C. Qualitative Examination for
Pathologic Organic Constituents
Gunnings Test
For Ketone bodies
Composed of 3 water-soluble molecules namely acetoacetate, beta-
hydroxybutyrate and acetone.
Source for energy in the citric cycle when glucose is insufficient.
The products of incomplete fat metabolism
Obtained from fasting or starving
Ketonuria
Reagents: Conc. Ammonium hydroxide, Lugols solution
Positive Control: 5ml Urine + 2ml acetone
Positive Results: Iodoform crystals
Principle: Ketone reacts with iodine, and the product of
that reaction reacts with hydroxide ions.
(IODOFROM)
Group Positive Control Urine Sample
#
1 Formation of yellow Iodoform Red-brown clear solution
crystals with precipitate
2 light yellow solution with white Yellow solution with
precipitate whitish precipitate
3 Light yellow solution with Golden solution with
precipitate precipitate
4 Yellow clear solution with Turbid solution with black
yellow precipitate precipitate
5
8 Colorless solution, with crystals Orange clear solution with
at the bottom white crystals at the
bottom
9 Iodoform crystals Red-orange solution
Group # Positive Control Urine Sample
1 Yellow precipitate Formation of clot in a
blue solution
2 Greenish blue solution Clear blue solution with
with brown precipitate light green precipitate
3 Green solution with Blue clear solution

precipitate at the bottom
4 Green solution with Blue solution with white
orange precipitate precipitate
5
8 Turbid blue green Clear blue solution
solution with yellow
precipatate
9 Muddy green solution Muddy orange solution
with precipitate with precipitate
Benedicts Test
For Glucose
A monosaccharide, aldohexose
Serve as an energy source
Usually there is little or no presence of glucose in the urine
Glucosuria
Reagents: Benedicts reagent
Positive Control: 1ml Urine + 1ml 1% glucose solution
Positive Results: Brick red precipitate
Principle: Reduction of cupric ions to cuprous form and
then converted to sugar acids. Cuprous ions combine with
OH- ions and form yellow cuprous hydroxide which
would convert to red cuprous oxide upon heating.
1 Yellow precipitate Formation of clot in a
blue solution
2 Greenish blue solution Clear blue solution with
with brown precipitate light green precipitate
3 Green solution with Blue clear solution

precipitate at the bottom
4 Green solution with Blue solution with white
orange precipitate precipitate
5
8 Turbid blue green Clear blue solution
solution with yellow
precipatate
9 Muddy green solution Muddy orange solution
with precipitate with precipitate
1 Turbid solution Light yellow clear
solution
2 Faintly clear yellow Faintly clear yellow
solution with white solution
precipitate
3 Turbid solution with Clear colorless
precipitate solution
4 White turbid solution Clear Colorless
with white precipitate solution
(cloudliness persistent)
5
8 Cloudy solution Clear colorless
solution
9 Cloudy solution Turbid solution with
precipitate
Extons Test
For Albumin
Globular protein made by the liver
Transports hormones, vitamins, drugs, etc.
This test can help determine if a patient has liver disease or kidney
disease, or if the body is not absorbing enough protein.
Albuminuria
Reagents: Extons reagent
Positive Control: 3ml Urine + pinch of albumin
Positive Results: Cloudiness
Principle: Negatively charged sulfosalicylic acid
neutralizes the positive charge on the proteins causing
denaturation, and hence precipitation of proteins upon
application of heat.
1 Turbid solution Light yellow clear
solution
2 Faintly clear yellow Faintly clear yellow
solution with white solution
precipitate
3 Turbid solution with Clear colorless
precipitate solution
4 White turbid solution Clear Colorless
with white precipitate solution
(cloudliness persistent)
5
8 Cloudy solution Clear colorless
solution
9 Cloudy solution Turbid solution with
precipitate
1 Brown solution on upper layer, Brown solution on upper layer,
orange solution on lower layer light yellow on lower layer
with green interphase
2 Red upper layer and bright Red upper layer and light
yellow lower layer yellow lower layer
3 Brown upper layer and light Brown upper layer, clear

brown lower layer, no colorless lower layer, no
interphase interphase
4 Emerald green interphase Colorless interphase
5
8 Dark red upper layer with green Dark red upper layer with
interphase, clear colorless lower yellow interphase, clear
layer colorless lower layer
9 Emerald green at point of Emerald green at point of
contact contact
Smiths Test
For Bile Pigments
Responsible for lipid catabolism
The two most important bile pigments are
bilirubin, which is orange or yellow, and its
oxidized form biliverdin, which is green.
Presence of Bilirubin
A yellow pigment in bile, a fluid made by the liver
Conjugated bilirubin and unconjugated bilirubin
Hyperbilirubinemia
Reagents: Tincture of alcoholic iodine mixture
Positive Control: 5ml Urine + 3-5 drops bile pigment
from pig gall bladder
Positive Results: Emerald green color at point of contact
Principle: Oxidation of the bile pigments to the colored
derivatives of the alcoholic iodide.
1 Brown solution on upper layer, Brown solution on upper layer,
orange solution on lower layer light yellow on lower layer
with green interphase
2 Red upper layer and bright Red upper layer and light
yellow lower layer yellow lower layer
3 Brown upper layer and light Brown upper layer, clear

brown lower layer, no colorless lower layer, no
interphase interphase
4 Emerald green interphase Colorless interphase
5
8 Dark red upper layer with green Dark red upper layer with
interphase, clear colorless lower yellow interphase, clear
layer colorless lower layer
9 Emerald green at point of Emerald green at point of
contact contact
1 Blue ring in a dark green Brown precipitate in
solution turbid solution
2 Formed blue ring until Light brown solution

whole solution turned with light brown
blue precipitate
3 Blue ring present in Brown upper layer in a
upper layer; brown turbid light brown
turbid blue lower layer solution with precipitate
on bottom
4 Blue ring in lower layer Brown lower layer
5
8 Brown solution with Brown turbid solution
brown upper layer with brown precipitate
9 Blue ring Blue ring

Test for occult blood

hidden blood
Hemolyzed: dissolved blood
Nonhemolyzed: intact red blood cells
Hematuria
Reagents: spatula of guaiac powder in 5 mL 95%
ethanol; hydrogen peroxide
Positive Control: 3ml acidified urine + 3 drops of blood
Positive Results: Blue ring
Principle: The peroxidase activity of the Heme which

decomposes and the liberated oxygen oxidizes the guaiac
powder.
1 Blue ring in a dark green Brown precipitate in
solution turbid solution
2 Formed blue ring until Light brown solution

whole solution turned with light brown
blue precipitate
3 Blue ring present in Brown upper layer in a
upper layer; brown turbid light brown
turbid blue lower layer solution with precipitate
on bottom
4 Blue ring in lower layer Brown lower layer
5
8 Brown solution with Brown turbid solution
brown upper layer with brown precipitate
9 Blue ring Blue ring

Conclusion
Urine samples from the 8 groups appeared to be what is
considered as normal urine, showing no abnormalities.
Urine samples tested with pH values ranging from 5 to 7.
Qualitative examination for normal organic constituents
showed that most urine samples contain Urea, Uric acid,
Indican and Creatinine.
Most urine samples were tested negative for the presence
of pathogenic constituents. However 1 urine sample
tested positive in all tests (Sample 9).
References
Fischbach, F., Dunning, M. B. III. A Manual of Laboratory and
Diagnostic Tests. Philadelphia: Williams & Wilkins, 2004.
Harr, R. R. Clinical Laboratory Science Review. Philadelphia:
F.A. Davis, 2007.
Goljan, E., Sloka K. Laboratory Testing in Clinical Medicine.
Sood, R. Concise Book of Medical Laboratory Technology. New
Delhi: Jaypee Brothers Medical Publishers. 2009.
Otten, R. Chemical Examination of Urine Part II: Protein,
Glucose.
http://www.slideshare.net/namarta28/urinalysis-methods-
observations-and-clinical-significance. Retrieved November
26,2016
http://edusanjalbiochemist.blogspot.com/2013/01/urinalysis-
chemical-examination.html. Retrieved November 26,2016

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Qualitative Examination of Urine

for Normal Organic Constituents

Solas, Karsten Sean

- Note the color, turbidity, acidity

B.1 Test for Urea

- Add 0.5 ml of 70% NaOH.

B.2 Test for Uric Acid

- Add 5 ml of 20% to the sample. Mix

B.3 Indican Test

- Add 5 ml of Obermayers reagent to the

B.4 Test for Creatinine

- Add 1ml of alkaline picrate solution to the

- Basify the 5 ml urine sample with 5 drops

- Add 5 ml Benedicts reagent to 8-10 drops of

- Mix 3 ml of the sample and 3 ml Extons

- Place 5 ml of urine sample in a test tube.

- Add 5 ml of 95% ethanol to a half spatula

1 6:30am Yellow Clear 6

1 6:30am Yellow Clear 6

B.1 Test for Urea

3 Bubble formation, clear yellow solution

4 Bubble formation, yellow clear solution

Test for Urea

B.1 Test for Urea

3 Bubble formation, clear yellow solution

4 Bubble formation, yellow clear solution

B.2 Test for Uric Acid

3 Light blue solution

4 Clear light blue solution

9 Blue solution ***

Test for Uric acid

B.2 Test for Uric Acid

3 Light blue solution

4 Clear light blue solution

9 Blue solution ***

B.3 Indican Test

9 Upper orange solution, bubbles formation on interphase,

B.3 Indican Test

9 Upper orange solution, bubbles formation on interphase,

B.4 Test for Creatinine

3 Clear Orange solution

4 Orange solution with precipitate

9 Orange solution on upper layer***

B.4 Test for Creatinine

3 Clear Orange solution

4 Orange solution with precipitate

9 Orange solution on upper layer***

3 Green solution with Blue clear solution

3 Green solution with Blue clear solution

3 Brown upper layer and light Brown upper layer, clear

3 Brown upper layer and light Brown upper layer, clear

2 Formed blue ring until Light brown solution

4 Blue ring in lower layer Brown lower layer

9 Blue ring Blue ring

Test for occult blood

Principle: The peroxidase activity of the Heme which

2 Formed blue ring until Light brown solution

4 Blue ring in lower layer Brown lower layer

9 Blue ring Blue ring

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