EXPERIMENT 10

ANALYSIS
OF URINE
URINE?
URINE CONSISTS OF:
Urine is the by product of all the activities of the
(96%) (4%)
kidney. A normal adult excretes about 600 to 2000 cc a
water dissolved solids:
day.
The volume varies on the volume of water intake, the
nature of the foods in the diet and the temperature of (2%) (2%)
the environment. The color or urine is due to the presence Urea: Other
of the pigment urochrome which gives its an amber (half) compounds
yellow color. Inorganic: Organic:
The normal consituents of urine are water, urea, sodium Cl-, Na, K. creatinine
chloride, creatinine, ammonia, dippuric acid and purine
trace amounts of: uric acid
bodies.
sulfate, HCO3 etc.)
The following pathological constituents are frequently
determined in a sample of urine: sugars, proteins, blood,
2
ketone bodies and bile.
 MATERIALS
Pipette Beaker 250 mL
Aspirators Stirring rod

REAGENTS
10 test tubes Litmus Paper
Test tube Bunsen Burner
Holder Tripod
Test tube rack Water Bath
Graduated
Cylinder

REAGENTS
5% Sucrose Concentrated HCl
2% acetic acid Picric Acid
Concentrated H2S04 (NH4)2SO4-NH3
10% NaOH 3% H202
Normal Urine Sample Benzidine in Glacial
Pathological Urine HAc
sample Sodium nitroprissude
Concentrated NaOH Benedicts reagents
Bromine Water 3
 OBJECTIVES
TO TEST FOR THE PRESENCE OF SOME NORMAL
PRODUCTS OF METABOLISM IN THE URINE

TO TEST FOR THE PRESENCE OF SOME

PATHOLOGICAL CONSTITUENTS OF URINE
A. PHYSICAL TEST OF URINE
Compare the characteristics listed below that are exhibited by normal urine and by pathological urine.

O B S E R VAT I O N
Properties Normal Urine Pathological Urine

1. Color LIGHT YELLOW AMBER YELLOW

2. Transparency Clear and TRANSPARENT WITH

transparent FLOATING PARTICLES
3. pH ACIDIC BASIC

4. Specific gravity 1.23 g/mL 1.1 g/mL

5. Odor PUNGENT ACETONE-LIKE

 O B S E R VAT I O N
NORMAL URINE PATHOLOGICAL URINE
B. TEST ON NORMAL
ORGANIC CONSTITUENTS

1. UREA
Place 10 drops of urine in a test tube, add 5 drops of concentrated NaOH then 2 drops of bromine
and water. Note the evolution of N2 gas.

2. URIC ACID
Place 5 mL of the urine in a beaker. Add 1 mL concentrated HCl and stir well and set aside until next
laboratory period. Describe the crystals that will be deposited on the sides of the beaker.

3. CREATININE: JAFFE’S PICRIC ACID REACTION

Place 10 drops of urine in a beaker, then add 5 drops saturated picric acid. Alkalinity with 10%
NaOH. A red color is produced which turns yellow when acidified.
 R E S U LT S
O B S E R VAT I O N A N A LY S I S
• UREA • when we add the concentrated NaOh and • The mixture of 10drops of urine, 5
the bromine water effeverscene of N2 gas drops of concentrated NaOH, and
can be seen at the bottom of the test tube. 2drops of bromine water will
produce effervescence in the test
tube. Thus, there is a presence of
urea in this test.
• URIC ACID • we added 1ml concentrated HCl to the • After stirring the mixture of 5mL
beaker with 5ml of urine, we stir it until urine and 1mL of concentrated HCl in
there is a few tiny white crystals forming at the beaker, the crystals are formed
the side of the beaker indicating the on the sides of the liquid. Since we
presence of uric acid. did not set aside the mixture for next
laboratory use, only the formation of
• CREATININE • after adding 5 drops of saturated picric crystals are analyzed.
acid in urine the color change from • Creatinine reacts with picric acid in
yellowish to orange then we added 10% of an alkaline solution it changes to
NAOH to the beaker with urine the color reddish color because of formation
became darker. of creatinine picrate
B. TEST ON NORMAL
ORGANIC CONSTITUENTS
UREA URIC ACID CREATININE
C . PAT H O L O G I C A L C O N S T I T U E N T S

Prepare 30 ml pathological sample of urine by adding

5 ml each of 5% glucose, 5% albumin, acetone, bile
and blood solution. Test the presence of the
Pathological Constituents.
 1

ACETONE: ROTHERA’S TEST

Place 5 mL of the Pathological Urine sample,
add 2.5 mL of (NH4)2SO4 – NH3 reagent and
2.5 mL of sodium nitroprusside reagent. Mix
and let it stand. Note the color produced.
 R E S U LT
A N A LY S I S CONCLUSION
a method of detecting acetone
and acetoacetic acid in urine. It The color of the 2- reagent is color white while the sodium
is performed by saturating the nitroprusside reagent is reddish brown and for the
sample with solid ammonium pathological urine sample is brown orange. When we
sulphate adding a few drops of added the 3 solutions together, the color became light
yellow brown and there were tiny particles floating at the
dilute sodium nitroprusside
top of the solution. After mixing the solution, change in color
solution, and then adding excess appeared wherein it became dark brown and also the
of concentrated ammonia. solution smells like acetone with vinegar. We’ve waited for
Development of purple color about 20 minutes to see, if the solution will change its color,
represents a positive result. after waiting we noticed that the color of the solution
became darker. Conclusion: In conclusion, the experiment
that we did turned out to be negative because the color of
the solution did not turn into purple colored complex.
Therefore, the solution has no acetone and acetoacetic
acid.
 2

GLUCOSE: BENEDICT’S TEST

Place 2.3 ml of Benedict’s reagent then 5 ml
of urine. Mix thoroughly. Boiled it in the water
bath for 2-3 minutes and allow it to cool by
itself. Take note of the color.
 GLUCOSE: BENEDICT ’S TEST
BEFORE AFTER

MIXING

BEFORE AFTER

BOILING
( 3 M I N S )
 R E S U LT
O B S E R VAT I O N C O N C L U S I O N A N A LY S I S
•Benedict’s solution can be used to test for the
Without mixing presence of glucose in urine. Glucose found to be
In our experiment, after
•The solution is gradient in color. present in urine is an indication of Diabetes
Yellow at the top and blue green at boiling, the solution turned mellitus. Once a reducing sugar is detected in
the bottom into avocado green in color. urine,further tests have to be undergone in order
Therefore, we conclude that to ascertain which sugar is present.The copper
With mixing: sulphate in Benedict's solution reacts with reducing
•The solution became pure green in there would be 0.1 to 0.5 sugars and the cupric ions to cuprous ions,these
color percent sugar in the are precipitated as red copper oxide, which is
After boiling: solution. insoluble in water (1). Alkaline medium is provided
•The solution became cloudy to the reaction by sodium carbonate present in
avocado green in color the reagent. The original colour of Benedict's
reagent is blue. It changes to green, yellow,
orange or red, according to the concentration of
glucose present in urine.
If the color upon boiling is changed into green,
then there would be 0.1 to 0.5 percent sugar in
solution. If it changes color to yellow, then 0.5 to 1
percent sugar is present. If it changes to orange,
then it means that 1 to 1.5 percent sugar is present.
If color changes to red,then 1.5 to 2.0 percent
sugar is present.
And if color changes to brick red,it means that
more than 2 percent sugar is present in solution.
 3

ALBUMIN: HELLER’S RING TEST

To 1.5 ml of concentrated HNO3 in a test tube,

deliver 1 ml of urine down the side of test tube.
Note down the observation.
ALBUMIN: HELLER’S RING TEST
BEFORE AFTER

MIXING
 R E S U LT
O B S E R VAT I O N CONCLUSION
Presence of fluffy zone at urine acid interface
Heller's test is a chemical test that shows
that strong acids cause the denaturation of
precipitated proteins. Concentrated nitric
acid is added to a protein solution from the
side of the test tube to form two layers. A
white ring appears between the two layers if Therefore, the result of our test using concentrated
the test is positive. Heller's test is commonly nitric acid and urine is positive. It’s pH indicates 1.0/100.
used to test for the presence of proteins so the Pathological urine that we used has presence of
in urine. protein.
 4

BLOOD: BENZEDINE TEST

Measure 3 ml of saturated solution of Benzidine
solution in a test tube. (Caution: Benzidine is
a mutagenic and carcinogenic). Add 3 ml of
3 % Hydrogen peroxide solution. Add about 3
ml urine. Note the changes.
BLOOD: BENZEDINE TEST
 R E S U LT
O B S E R VAT I O N & C O N C L U S I O N
A sensitive test for the presence of blood based on
the production of a blue color upon constancy with
a solution of benzidine, hydrogen peroxide and
glacial acetic acid.

A root-beer like formed in the upper part of the fuid

then the bluish cloud like stayed at the center then a
blurrish liquid sat still at the bottom.

(Light to dark red)

 5

BILE PIGMENT: GMELIN’S TEST

Place 1 ml of concentrated HNO3 in a test
tube. By means of pipette, deliver down
the side of the tube 5 ml of urine. Do not
shake and note the color of the rings.
 R E S U LT S
O B S E R VAT I O N CONCLUSION
Different colored rings between the two
layers are visible if bile pigments are
present as they are oxidized to various
chemical products. Nitric acid is used as
the oxidizing agent. Blue, green and violet
rings are seen if bilirubin is present. Gmelin's
test is not sensitive so a positive result
always indicates the presence of bile
pigments but a negative result does not
exclude the presence of small quantities of
bile pigments.
 6

BILE ACIDS & SALTS: PETTENKOFER’S TEST

Place 20 drops of urine, in a test tube, then add 1
drop of 5% sucrose. Let 1 ml concentrated H2SO4
slide at the side of the test tube. Do not shake yet.
A red ring develops at the point of contact of the
two solutions. Don't make mistake the brown color
resulting form the charring of sucrose by H2SO4 for
the red ring. Stir the mixture and note the effect.
BILE ACIDS & SALTS: PETTENKOFER’S TEST

BEFORE AFTER

MIXING
 R E S U LT
O B S E R VAT I O N CONCLUSION
Result: (+) Formation of red color at the point of
contact. CONCLUSION: Upon adding 1 drop of
5% sucrose in 20 drops of pathological urine, we
noticed that the top part of the solution
produced a cloudy green colored substance
and by adding the concentrated H2SO4 the
whole solution became a bright cherry-red
colored solution. Thus, this red reaction which
was formed at the point of contact shows the
presence of bile acids. Since, it was proven by
Pettenkofer that sucrose can be converted to
fat in vitro; bile acid took its action. Thus, the
addition of the chemical sulphuric acid shows
the positive result on the bile acid’s presence by
the formation of a red color.
 A 24-hour urine collection helps diagnose kidney problems. It is often
done to see how much creatinine clears through the kidneys. It’s also
Why must a 24-period done to measure protein, hormones, minerals, and other chemical
sample of urine be compounds.
used for examination if The first voided morning specimen is particularly valuable because it is
more concentrated and abnormalities are easier to detect. An early
its detailed
morning specimen is also relatively free of dietary influences and changes
composition has to be
due to physical activity. In collecting any urine specimen, it is always
determined? For a
important for the nurse to observe specific agency protocols, to check
simple routine
with the laboratory regarding the need for refrigeration or preservation of
qualitative analysis?
specimens, and to follow universal precautions. Single random specimens
Why is an early
may be taken at any time of the day or night. Timed specimens range
morning sample of
from short-term 2-hour collections to 24-hour collections.
urine used for test and
A 24-hour urine specimen is an extremely important diagnostic test
not the urine after
because it reveals how the kidney adjusts to changing physiologic needs
meal? over a long period. Substances excreted by the kidney are not excreted
at the same rate or in the same amounts during different periods of day
and night; therefore, a random urine specimen does not accurately
represent the processes taking place over a 24-hour period. However, a
24-hour urine specimen is useful only when all the patient's urine is
collected for 24 hours. Even if just one sample is discarded, the results will
be inaccurate. The nurse must ensure that the patient and all assistive
personnel understand the importance of saving all the urine. To begin the
24-hour collection, the person voids and discards the urine already in the
bladder. All urine starting with the next voiding is collected for the next 24
hours and put into a large collection bottle. To prevent breakdown of 27
urinary components, the collection has a preservative added to it or is
refrigerated.
28
INTERPRETATION OF URINE APPEARANCE
Clear or straw-colored is normal. Colour varies with hydration
status and urine concentration

Hematuria, hemoglobinuria, myoglobinuria. Menstrual blood

contamination, foods such as beetroot, drugs such as Rifampicin.

Bile pigments, myoglobin, drugs. Bowel-bladder fistula.

Green-blue – Pseudomonas UTI

Cloudy-infection, ejaculate or vaginal discharge contamination.

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YOU