ANALYSIS OF NORMAL URINE

Sub-competency: BI 11.4- Perform urine analysis to estimate and determine normal and
abnormal constituents

Physical characteristics:

The quantity of urine excreted by normal subjects ranges from 1000 – 2000 ml per day. The
volume is influenced by intake of fluid, proteins and sodium chloride. Excessive perspiration
and strenuous exercise decrease the volume of urine. Specific gravity of normal urine varies
in the range of 1.012 – 1.024. It can be as low as 1.001 when water intake is high and as high
as 1.04 when volume of urine is low. Specific gravity is directly proportional to the
concentration of solutes excreted. Normal pH of urine ranges from 4.8 – 7.5 with an average
of 6.0. High protein diet gives rise to a more acidic urine. Urine becomes slightly alkaline
when the diet is rich in vegetables and fruits. Normal urine is pale yellow. When the output
of urine is low it appears deep yellow. Freshly voided urine is clear and transparent. On
standing it may become turbid due to precipitation of phosphates.

Chemical characteristics:

Normal urine contains both inorganic and organic constituents. The inorganic constituents

include Na+, K+, Ca++, Mg++, NH4 +, CI–, H2 PO4 –, HPO4– , SO4 –, and traces of HCO3
ions. Normal organic constituents are urea, uric acid and creatinine. The total non-protein
nitrogen varies from 10 – 15 gm/day depending mainly on the protein intake. In addition to
these major organic constituents, detoxified products like indican and ethereal sulphates are
found in urine. Routine analysis of urine includes tests for Cl, SO 2-, phosphate, Ca2+, NH4
+, urea, uric acid, creatinine, ethereal sulphate and urobilinogen.

Test Observation Inference

Tests for inorganic constituents:

Chloride: Curdy white Chloride is precipitated as silver

precipitate chloride with AgNO3 in
2 ml urine + 0.5 ml of
presence of HNO3
concentrated HNO3 + 1 ml
AgNO3

Note: On an average diet 24-hour urine contains 8-15 g of sodium chloride.

Sulphate: White Sulphate is precipitated as

precipitate barium sulphate with barium
2 ml urine + 2 ml BaCI2 chloride.

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Note: Sulphate is derived from the catabolism of sulphur containing amino acids. On
an average diet about one gram of sulphate is excreted per day. About 85% – 95% of
sulphur is excreted as inorganic sulphate.

Inorganic phosphate: Yellow color Inorganic phosphate is

or precipitate precipitated as canary
5 ml urine + few drops of (A green color yellow ammonium
concentrated HNO3 + a pinch indicates that phosphomolybdate.
of ammonium molybdate. nitric acid is
Warm insufficient).

Note: About 1 g. of phosphate is excreted per day.

Ammonia: Red litmus Ammonia liberated turns

turns red litmus blue.
5 ml urine + 2% Na2CO3
(till the red litmus turns Blue
blue), boil. During boiling,
hold a piece of

Moistened red litmus paper

at the mouth of the test tube.

Note: Urinary ammonia is derived from glutamine and other amino acids in kidney.
The normal daily output of ammonia is about 0.5 – 0.8 g. There is an increase in
ammonia excretion when acid forming foods are taken.

Test Observation Inference

Tests for organic constituents:

Urea:

Sodium hypobromite test: 2 Brisk Hypobromite decomposes

ml of urine + 4 – 5 drops of effervescence urea to N2 gas.
sodium hypobromite of nitrogen gas
solution

H2NCONH2 + 3NaOBr + 2NaOH N2 + Na2CO3+ 3NaBr + 3 H2O

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Specific urease test: Urease converts urea
into ammonia and carbonic
Label two test tubes as 'test' Pink color in acid. Under the pH of the
and 'control'. test reaction condition NH3 &
5 ml urine + a drop of carbonic acid are converted
phenolphthalein. (Since to ammonium carbonate.
normal urine is acidic pH goes above 9.5.
phenolphthalein does not
No pink color Since the contents are
give any color) + 2%
in control alkaline phenolphthalein
Na2CO3 drop by drop till a
gives pink color.
pink color is seen. At this
stage pH is > 8.5. Add 1%
acetic acid carefully till
the pink color is just' Urea is hydrolyzed by
discharged. pH at this stage Pink color in urease to form ammonium
is 8.3. Add to the contents test carbonate which makes the
solution alkaline.
Test: 2 ml of well mixed
urease suspension.

Control: 2ml urease

suspension which has been
heated strongly Mix,
incubate at room
temperature for 15 min.

Urease

NH2 CONH2 + 2H2O H2CO3 + NH3 (NH4)2 CO3

Note: Urea is formed in liver as the end product of protein catabolism and so its

excretion depends on protein intake. About 20 – 30 g. urea are excreted in 24 hours.

Test Observation Inference

Uric acid: Blue color Uric acid is a reducing agent.

Phosphotungstic acid In alkaline condition it

reduction test: reduces phosphotungsticacid
to tungsten blue.
2 ml of urine + few drops of
Phosphotungstic acid reagent +

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few drops of 20% Na2CO3

Schiff’s test:

Wet a piece of filter paper with Black color is Uric acid reduces ammoniacal
few drops of ammoniacal formed after some AgNO3 to metallic silver which
AgNO3 solution. Add 1 or 2 time is black in color.
d r o p s o f urine solution on the
same paper.

Note: Uric acid is the end product of purine catabolism. The daily output of uric acid
varies in the range of 0.6 – 1 g.

Creatinine:

Jaffe's test: label 2 test tubes as Orange color in Creatinine reacts with alkaline
'test' and 'control'. Into test, 2 ml ‘test’. Yellow picrate solution to form orange
urine solution + 2 ml of saturated color in 'control' creatinine picrate.
picric acid solution + few drops
of 10% NaOH.

Into 'control' : 2 ml

H2O + 2 ml of picric acid + 10%

NaOH

Note: Urinary creatinine is derived from muscle creatine. Daily excretion is fairly
constant in an individual. It depends on the muscle mass. Excretion in adult male is
about 2 g/day and 1 g/day in females.

Test for Urobilinogen: 5 ml Red color is Urobilinogen reacts with P-

of freshly voided urine + 1 seen when dimethyl amino
ml of Ehrlich reagent. Mix. viewed benzaldehyde to give a red
Let it stand for 5 minutes. through the color.
mouth of the
test tube

Note:Normal urine contains traces of urobilinogen. This is derived from bilirubin by

the action of bacterial flora in the intestine, enters into circulation and then excreted
by the kidneys. Absence of urobilinogen in urine is suggestive of obstructive jaundice.
Excess of urobilinogen in urine is indicative of hemolytic jaundice.

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Determination of specific gravity:

This is done with a urinometer (a hydrometer) consists of a thin stem graduated from 1000 to
1060 corresponding to specific gravities of 1.0 to 1.06. The bulb at the bottom is suitably
weighted. Urinometer is calibrated at 60°F (15°C).

Take sufficient urine in a container. Allow the urinometer to float in urine without touching
the sides. Observe the specific gravity reading corresponding to the meniscus of urine. Note
the temperature of urine.

Suppose the meniscus of the urine coincides with the reading 1010 and temperature of urine
is 37°C. Since the urine is at a higher temperature than the temperature of calibration of
urinometer, a temperature correction has to be applied.

Correction is: For every 3°C rise over the temperature of calibration, a correction factor of
'one' is added to the observed reading. The difference between 37 and 15 is 22°C. This when
divided by 3 gives = 7.

Corrected specific gravity = 1010 + 7

= 1.017

Applying the decimal point, the specific gravity of the given urine is close to 1.017.

Fig.: Urinometer (an apparatus to determine specific gravity)

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Observations and Calculations

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Sub-competency: BI 11.4- Perform urine analysis to estimate and determine normal and
abnormal constituents

Attempt 1 Attempt 2 Attempt 3

B M E B M E B M E

Test for chloride

Test for sulphate

Test for inorganic

phosphate

Test for ammonia

Sodium hypobromite
test

Specific urease test

Phosphotungstic acid
reduction test

Schiffs test

Test for creatinine

Grade

Signature of the
assessor with date

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