ANALYSIS of NORMAL URINE - Writing Material For Notebook

Experiment No.
Date:
Aim: Physical and Chemical analysis of Normal Urine sample
URINE ANALYSIS
• Urine is an excretory product of the body and presence of certain substances in the urine reflects
the metabolic state of the body.
• Since it can be easily collected and examined, routine and microscopic examination of urine is
an important preliminary step in the diagnosis of various pathological conditions.
COLLECTION OF SPECIMENS AND ITS PRESERVATION
Like all biological specimens, urine has to be collected and adequately preserved to prevent
contamination and bacterial overgrowth since it is a very good culture medium.
The following methods can be used to collect a urine specimen:
• Clean catch midstream urine sample: External genitalia are cleaned, and first 10 ml of urine is
voided. The midstream portion of the urine is collected in a sterile container for testing. This
sample is best for bacteriological study of the bladder and kidney.
• Catheter specimen of urine: A catheter is a thin rubber tube that's inserted through the urethra
into the bladder. It is used for microbiological examination in critically ill patients or in urinary
tract obstruction, only.
• Suprapubic aspiration: The urine is aspirated from the bladder using a 22-gauge needle
following aseptic technique. This technique is used to collect a sterile urine sample in infants
and as an alternative to catheterization.
The first voided morning urine, which is the most concentrated, is best for routine urinalysis
Depending on the analyte to be tested the timing of urine specimen collection can vary as:
1. Random sample: The sample is collected on the spot at any time of the day. It has limited utility
and is used for detection of glucose, ketone bodies and proteins.
2. Timed sample:
• Early morning/ mid streamed morning sample: Specimen is most concentrated and preferred
for microscopic examination for bacteria and for detection of abnormal amounts of constituents
• 24-hour urine sample: For certain analytes such as steroid hormones which show diurnal
variation.
• The patient is asked to discard the first voided urine and start collection of all the urine samples
voided subsequently till the first urine sample of the next morning in a sterile container.
URINE SAMPLE PRESERVATION
• Ideally urine sample should be analyzed within 2 hours of collection.

• If analysis within 2 hours is not possible, most satisfactory form of preservation is refrigeration
at 4°C.
• Chemical preservatives like Sodium benzoate or Boric acid should be used if sample is
preserved at room temperature.
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URINE ANALYSIS
PHYSICAL MICROSCOPIC
CHEMICAL EXAMINATION
EXAMINATION EXAMINATION
1) Colour 1) Cells
A. Normal constituents: 2) Crystals
2) Appearance Inorganic & Organic
3) Odour 3) Casts
B. Abnormal
4) Specific gravity constituents
5) Volume 1) Proteins
6) pH 2) Glucose
3) Ketone Bodies
4) Blood
5) Bilirubin
6) Urobilinogen
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Objective A: Physical Examination of normal urine sample

S.No Physical Properties Observation
1. pH
2. Volume
3. Appearance
4. Colour
5. Odor
6. Specific Gravity
Objective B: Chemical Examination of normal urine sample.
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Normal urine contains 90-95% of water and about 60g/day of solid material, comprising of organic and
inorganic substances.
Organic Substances Volume

Urea 25-30 g/day
Uric acid 0.5-0.8 g/day
Creatinine 1-1.5 g/day
Hippuric acid 0.7-0.8 g/day
Inorganic Substances Volume

Chloride 10-15 g/day
Sodium 3-5 g/day
Potassium 2-2.5 g/day
Calcium 0.1-0.3 g/day
Phosphates 0.8-1.3 g/day
Sulphates 1-1.2 g/day
Ammonia 0.7-0.8 g/day
A. Tests for inorganic constituents present in the urine (make this table
on lined page)
EXPERIMENT OBSERVATION INFERENCE
1. Test for Chloride Curdy white precipitate of Chloride present

silver chloride seen
2 ml urine sample + 0.5 ml conc.
HNO3 Followed by 1ml AgNO3
White precipitate is
2. Test for inorganic sulphate
observed
Sulphate is present
1ml of sample + 1ml 10% BaCl2
3. Test for Calcium White precipitate of calcium Calcium present

oxalate formed
2 ml of sample+ 5 drops of 1% acetic
acid + 2ml potassium oxalate
4. Test for inorganic Canary yellow precipitate

phosphorous formed
Inorganic phosphorous
3 ml of sample + 5 drops conc. HNO3 present
+ pinch of ammonium molybdate &
heat it
5. Test for ammonia Litmus paper turns blue Ammonia present

2 ml of sample + 200 microlitre drops
of 2% Na2CO3 till solution is
alkaline. Boil the test tube & hold the
piece of litmus paper at the mouth of
it.
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1.Test for Chloride
Principle: Chloride present in urine is precipitated as silver chloride (AgCl) in the presence of conc.
HNO3
2. Test For Sulphate
Sulphate is derived from catabolism of sulphur containing amino acid (cysteine, methionine)
85-95% of sulphur is excreted as inorganic sulphate and rest as ethereal sulphate (organic sulphate).
Principle: Inorganic Sulphate is precipitated as barium sulphate in presence of barium chloride
3. Test For Calcium
Principle: With potassium oxalate in acidic conditions, calcium is precipitated as calcium oxalate
4. Test for inorganic phosphorous
Principle: Upon warming with ammonium molybdate in the presence of HNO3, inorganic phosphate
is precipitated as canary yellow ammonium molybdate
5. Test for Urinary ammonia
Urinary ammonia is derived from glutamine and other amino acids in the kidneys.
Principle: When Na2CO3 is added to the urine ammonia is releases which turns red litmus into blue
when it is brought close to the mouth of the test tube
B. Tests for organic constituents present in the normal Urine (Table on
lined page)
EXPERIMENT OBSERVATION INFERENCE
1. Test for urea Brisk effervescence of N2 Urea is present

gas is seen
2 ml of sample +4 drops of
sodium hypobromite
solution
2. Test for uric acid Pinkish coloured solution is Uric acid is present
seen
0.5ml uric acid reagent+10
microlitre diluted alkaline
urine sample
3. Test for Creatinine: reddish brown colour turns Creatinine is present.

yellow after adding NaOH
a. Nitroprusside test: 2 ml
of sample + 0.5 ml sodium
nitroprusside+ make
solution alkaline by adding
with 0.5 ml of 10% NaOH
wait for 1 min
b. Jaffe’s (Picric acid) test Orange coloured solution of Creatinine is present.

creatinine picrate is formed.
2 ml of sample + 2ml
saturated picric acid + few
drops of 10% NaOH
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1. Test For Urea:
Principle: Sodium hypobromite decomposes urea to release N2 gas
Urea+ Sodium hypobromite + NaOH N2 gas+ Na2CO3+NaBr+H2O
2. Test for uric acid: (a)modified trinder method
Principle:
• Uric acid is oxidised to allantoin by uricase with the production of H2 O2

• The peroxide reacts with 4-aminoantipyrine (4-AAP) and DHBS in the presence of peroxidase
to yield a quinoneimine dye. The absorbance of this dye at 505 nm is proportional to uric acid
concentration in the sample. Color intensity of Quinoneimine directly propotional to
concentration of uric acid
The series of reactions involved in the assay system is as follows:
UA+O2+H2O URICASE Allantoin+CO2+ H2O2
H2O2+ 4 AAP+TBHB PEROXIDASE Quinoneimine (Chromogen) +4H2O

4 AAP-4-aminoantipyrine
TBHB- 2,4,6-Tribromo-3-hydroxybenzoic acid
3. Test For Creatinine:

a. Nitroprusside test
Principle: Creatinine have reducing property it reduces sodium nitroprusside in alkaline medium
& gives reddish brown color.
b. Jaffe’s (Picric acid) test
Principle: Under alkaline conditions creatinine reacts with picric acid solution to form an orange
red solution of creatinine picrate
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Result:
In the provided urine sample, organic constituents such as Urea, Uric acid and creatinine is present &
inorganic constituents’ chloride, sulphate & calcium are also present.

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Experiment No.

Aim: Physical and Chemical analysis of Normal Urine sample

• Ideally urine sample should be analyzed within 2 hours of collection.

Objective A: Physical Examination of normal urine sample

Objective B: Chemical Examination of normal urine sample.

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Organic Substances Volume

Inorganic Substances Volume

1. Test for Chloride Curdy white precipitate of Chloride present

3. Test for Calcium White precipitate of calcium Calcium present

4. Test for inorganic Canary yellow precipitate

5. Test for ammonia Litmus paper turns blue Ammonia present

1. Test for urea Brisk effervescence of N2 Urea is present

3. Test for Creatinine: reddish brown colour turns Creatinine is present.

b. Jaffe’s (Picric acid) test Orange coloured solution of Creatinine is present.

(Write Principle on opposite blank page of file)

• Uric acid is oxidised to allantoin by uricase with the production of H2 O2

H2O2+ 4 AAP+TBHB PEROXIDASE Quinoneimine (Chromogen) +4H2O

3. Test For Creatinine:

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