QUALITATIVE ANALYSIS OF ABNORMAL

URINE

Ex.No:

DATE
CONSTITUENT
GENERAL PROCEDURE FOR ANALYSIS OF NORMAL

OF URINE

INFERENCE
S.NO. EXPERIMENT OBSERVATION

Physical Examination: Normal urine is clear

Clear
a) and transparent.
Due to the presence of
Straw coloured
Appearance urochrome.

PH of the normal urine

6
b)Colour is 4.8-7.0

1.018
c)PH Normal specific
gravity of urine is

d)Specific gravity 1.016-1.022

| 2. Test for inorganic constituent

a) Test for chloride:
To the 5ml of urine sample,
A white precipitate of silver Presence of chloride
add con. nitric acid and 1ml of chloride is formed. in urine

3% silver nitrate.

b)Test for calcium and

Phosphates:
Take 10m of urine add 2ml
of ammonium hydroxide boil it
White precipitate is formed.
& cool, then flter it. Filtrate is

40 P a ge
 collected and the precipitate is Presence of calcium.
dissolved in quantity of acetic Yellow colour precipitate
acid and divided into 2 parts. occurs. Presenceof
i) To one part, add 1ml of 5%| phosphate.
potassium oxalate solution.

i) To the 2nd part, add 1ml

con. nitric acid if necessary

boil and add 1ml of

ammonium molybdate reagent Phenolphthalein indicator in Presence of ammonia.

and mix well.
the glass rod changes to

c) Test for ammonia: pink colour.

Take 5ml of urine in a test

tube & add a drop of
phenolphthalein and add 2%|
sodium carbonate drop by
drop until the colour becomes
faint pink. Allow to boil and A white precipitate of Presence ofinorganic

hold a glass rod dipped in sulphate.

Darium sulphate is formed.
phenolphthalein at the mouth |
of the test tube.

sulphate:
d) Test for inorganic
test
Take 5ml of urine in a

tube. Add 1ml of con.

Hydrochloric acid. Mix welland

add 5ml of 10% barium
chloride solution. Filter it and
next
use the filtrate for

experiment.

of
Precipitate is formed. Due to the presence
constituent:
Test for organic
3
41|Page
 organic constituent.
Collect the above filtrate &

allow it to boil.

4. Test for urea:

Effervescence is produced. Alkaline hypobromite
Take 5ml of urine in a test| react with urea
which

tube & add 2 drops of freshly forms gases like

prepared alkaline sodium nitrogen & co2
hypobromite solution.

5. Test for creatinine:

Jaffe's test
3ml of urine and 3ml of water
red colour is Due to the formation
are taken in 2 separate test Orange
developed in the test tube of creatinine picrate
tubes. 1ml of saturated picric
containing urine.
acid and 10 drops of 10%
sodium hydroxide are added to
both the test tubes and mixed.
Wait for 5 minutes.

6 Test for uric acid:

Blue colour is appeared. Presence of uric acid.
Take 3ml of urine and add
1ml of 1% sodium carbonate
and 1ml of dil.

Phosphotungstic acid.

42 P a ge
 REPORT:
The constituents of normal urine are

Chloride

Calcium

Phosphate
Ammonia

Inorganic sulphate
Organic constituent

Urea

Creatinine

Uric acid

43|Page
 Ex.No:

DATE
GENERAL PROCEDURE FOR ANALYSIS OF ABNORMAL

cONSTITUENT OF URINE

(Pathological constituents of urine)

The commonly encountered pathological chemical constituents of urine are

1. PROTEIN: May be albumin or globulin

2. BLOOD: Haemoglobin, Erythrocytes
in special lactose, galactose, pentose
3. REDUCING SUGAR: Usually glucose and cases

and rarely fructose

4. KETONE BODIES: Acetone, aceto acetic acid

5. BILE SALTS & BILE PIGMENTS: Sodium and potassium salts of glycol/ taurcholic
acids,Bilirubin
6. PORPHOBILINOGEN

7.UROBILINOGEN

OBSERVATION INFERENCE
S.NO. EXPERIMENT
TESTFOR PROTEIN: Presence of protein
1.
Coagulation occurs
a)Heat coagulation test:

Fill 4 of a test tube with the

urine acidified with 2% acetic

acid mix & heat the upper portion.

Principle: Urine contains mainly albumin which is a heat coagulable protein

b) Sulphosalicylic acid test:

To 5ml of urine, add 1ml of

White precipitate is Presence of protein
formed
20% Sulpho salicylic acid.

the
Principle: Protein is precipitated by Sulpho salicylic acid by removal of charges on
protein

c)Heller's test: A white ring of meta Presence of protein

Take 3ml of con. Nitric acid and proteins appears at the

44 P age
 add 2ml of urine alorng the sides Junction of thes lhds

of test tube.

2. TEST FOR BLOOD:

a)Ortho-Tolidine test:
To 6 drops of freshly preparodAransienl darktret hastnohan
0-Tolidine add 6 drops of our is lormrt

hydrogen peroxide and 4 drops of

previously boiled urine.

Principle:
Heme part of haemoglobin has perozidisa ikes activity wtich releas thhs nass1i
oxygen from H202. This nascent oxygen ozidizes 0 Tolidine vhihi ty ss tye4

colour. Colour disappears after few minutes.

b)Benzidine test
Presefirs
To 6 drops of freshly prepared
of A transient dark green fissoir
benzlidine add 6 drops
hydrogen peroxide and 4 drops of colouris formed
previously boiled urine

3. TEST FOR REDUCING SUGAR:

Colour dhanges fron Preserica reutinng
Benedict's test:
blue to green, yellou, sugar.
To 5ml of Benedict's reagent,
orange or brick red
add 8 drops of urine sample. Boil preapitate.
over a flame for 2 minutes or

in boiling water bath for

place a

three minutes and allow to cool.

Principle:
cartonate and vium
Benedict's reagents contain copper sulphate, sdiun
citrate. The mild alkali sodium carbonate converts gucose into enedoi. This eredr

that is unstabie and dempnes on

reduces copper sulphate to cuprous hydroside
shades f
cuprous onide wil rave dMerent
boiling to cuprous oxide. The precipitated
colour depending upon the
concentration.

45 Page
 as
of cupric 1on
Sodium citrate present in this reagent
prevents the precipitation
self life
citrate complex. It
also improves the
sodium
Cupric hydroxide by forming cupric
carbonate and copper
interaction between sodium
of the reagent by preventing a n

sulphate.

4. TEST FOR KETONE BODIES:

a)Rother's test:
Take 5ml of urine and fully Presence of acetone
A permanganate colour
saturated with solid ammonium
appears.
sulphate. This is to remove
substances, which may interfere

with the test. Then, add5 drops of

a freshly prepared solution of
sodium nitro prusside and gently|
shake. Then add a few ml of

con.ammonia and mix it.

Principle
Ammonia) react with ketone
The nitro prusside in alkaline medium(due to con.

colour.
group to form a permanganate
Presence of aceto acetic
b)Gerhardt's test.
A wine red colour
acid
To about 5ml urine in a test
appear
tube, add drop wise 10% ferric

chloride

TEST FOR BILE SALTS & BILE

5
PIGMENTS:

a)Hay's test: (for bile salt) Test tube (A) Presence of bilesalts

Take two test tubes one with containing sulphur

5ml urine (A) and other with 5ml powder sink.

of water (B). Now, gently sprinkle

flowers of sulphur into both.

Principle:
Hay's test is based on the fact that bile salts lower the surface tension of urine

46 Page
 allowing the sulphur to sink.

b) Fouchet's test (For bile

Green colour appear Presence of bile
pigments)
Pigments
Take 5ml of urine add a few

crystals of magnesium sulphate

and shake the tube till it
dissolves. Now add 10% barium

chloride in excess (about 10ml).

A precipitate of barium sulphate
is formed. The bile pigment get
adsorbed to the precipitate of
barium sulphate. Filter the

contents of the tubes, the filter

be discarded. Dry the
may

precipitate by using filter paper.

To the dry precipitate add a drop
contain
offouchets reagent that
ferric chlorides as the oxidising

agent

Principle: bilirubin to
biliverdin
it oxidises
a oxidizing agent
Ferric chloride
reagent act as

(blue)
(green) or bilicyanin
of
TEST FOR UROBILINOGEN Presence
Chloroform layer
6.
A N D P O R P H O B I L I N O G E N :

changes to pink Urobilinogen

Ehrlich's diazo test: colour

To 5ml of
urine add
5ml of of
Presence
ehrlichs diazo
reagent mix well
Porphobilinogen.

to stand for 10 Aqueous layer

allow it
and
sodium changes to pink
mts.Add 5ml of saturated
colour

47 P age
 acetate and mix. Now add 5ml of
chloroform and shake vigorously
allow the
for a few seconds and

layers to separate.

48 | P a ge
 EX.NO:

DATE:

QUALITATIVE ANALYSIS OF ABNORMAL

cONSTITUENT OF URINE
SAMPLE- I
S.NO. EXPERIMENT
1 Test For Protein:
OBSERVATION INFERENCE
a)Heat coagulation test Presence of protein
Coagulation occurs
Fill of a
test tube with the
urine acidified with 2%
acetic
acid mix & heat the
upper
portion.

b) Sulphosalicylic acid test:

To 5ml of urine add 1ml of White precipitate is Presence of protein
20% Sulpho salicylic acid. formed
c)Heller's test A white ring of meta Presence of protein
Take 3ml of con. nitric acid add proteins appears at the
2ml of urine along the sides of junction of the fluids.
test tube.

2. Test for blood:

a)Ortho-Tolidine test: Absence

To 6 drops of freshly prepared
Notransient dark
colour formed haemoglobin
0-Tolidine add 6 drops of | green is
hydrogen peroxide and 4 drops

of
previously boiled urine.
b)Benzidine test Absence of
prepared
To 6 drops of freshly No transient dark
haemoglobin
benzlidine add 6 drops of
formed
and 4 drops 9reen colouris
hydrogen peroxide

of
previously boiled urine.

9 |P agee
 . Test for reducing sugar:
Benedict's test: Colour changes from Presence of reducing

To 5ml of Benedict's blue to orange sugar

reagent
add 8 drops of urine sample. Boil precipitate.
over a flame for 2 minutes or

place them in a boiling water

bath for three minutes and allow
to

COol
Test for ketone bodies:
a)Rother's test:
Take 5ml of urine and fully Absence of acetone
No
saturated with solid ammonium
permanganat
sulphate. This is to remove
e colour appear
substances, which may interfere
with the test. Then, add5 drops of
a freshly prepared solution of
sodium nitro prusside and gently
shake. Then add a few ml of
con.ammonia and mix it.

b)Gerhardt's test:
No wine red colour Absence of aceto acetic
To about 5ml urine in a test
appears. acid
tube, add drop wise 10% ferric
chloride

5. Test for bile salts & bile

pigments:
a)Hay's test: (for bile salt) Test tube (A) Absence of bilesalts
Take two test tubes one with containing sulphur
5ml urine (A) and other with 5m powder does not sink.

water (B). Now gently sprinkle

flowers of sulphur into both.

50 P age
 b) Fouchet's test (For bile
No colour Absence of bile
pigments)
Gree pigments
Take 5ml of urine add a few
n appears.
crystals of magnesium sulphate
and shake the tube till it
dissolves. Now add 10% barium
chloride in excess (about 10ml).
A precipitate of barum sulphate
is formed. The bile pigments get
adsorbed to the precipitate of
barium sulphate. Filter the
contents of the tubes, the filter

may be discarded. Dry the

precipitate by using filter paper.

To the dry precipitate add a

drop of fouchets reagent that

contains ferric chlorides aas

oxidising the

agent.

for Urobilinogen and

6. Test
No Chloroform layer Absence
porphobilinogen:

Ehrlich's diazo test: changes to pink Urobilinogen

To 5ml of urine add 5ml of colour

ehrlichs diazo reagent mix well

Absence o
for 10 mts.
and allow it to stand
saturated sodium No Aqueous layer Porphobilinogen
Add 5ml of
changes to pink
acetate and mix.
Now add 5ml
colour
Shake vigorously
of chloroform.
allow the
for a few seconds and

layers to separate.

51 |P a ge
 REPORT:

The given unknown sample contain

1. Protein

2. Reducing sugar

52| P age
 EX.NO:

DATE:
ABNORMAL CONSTITUENT OF URINE SAMPLE- II
QUALITATIVE ANALYSIS OF
OBSERVATION INFERENCE
S.NO. EXPERIMENT
bsence of protein
Test For Protein:
No Coagulation occursS
a)Heat coagulation test:

Fill 4 of a test tube with the

urine acidified with 2% acetic

acid mix & heat the upper

portion.

test:
b) Sulphosalicylic acid Absence of protein
No White precipitate is
To 5ml of urine add 1ml of

20% Sulpho salicylic acid. formed

of meta | Absence of protein

No white ring
c)Heller's test
proteins appears at the|
Take 3ml of con. nitric acid add
junction of the fluids.
2ml of urine along the sides of
test tube.

Test for blood:

2.
a)Ortho-Tolidine test:
absence of haemoglobin
Notransient dark
green
prepared
To 6 drops of freshly
O-Tolidine add 6 drops of colour is formed

hydrogen peroxide
and 4 drops
of
previously boiled urine.
b)Benzidine test: Absence of haemoglobin
prepared
To 6 drops of freshly
Notransient dark
green
6 drops of
benzlidine add
formed
drops colour is
peroxide and 4
hydrogen
boiled urine.
of previously

53 | P age
 of reducing
3. Test for reducing sugar: from
Presence
Colour changes
Benedict's test: orange sugar
blue to
To 5ml of Benedict's reagent
precipitate.
add 8 drops of urine sample. Boil
over a flame for 2 minutes or

place in a boiling water bath for

three minutes and allow to cool.

4. Test for ketone bodies:

a)Rother's test:

Take 5ml of urine and fully Absence of acetone

No
saturated with solid ammonium
permanganat
sulphate. This is to remove
e colour appear
substances, which may interfere
with the test. Then, add5 drops
solution of
of a freshly prepared
sodium nitro prusside and gently
shake. Then add a few ml of

con.ammonia and mix it.

b)Gerharde'stest: No wine red colour

Absence of aceto acetic

To about 5ml of urine in a test

acid
appears.

tube, add drop wise 10% ferric

chloride

5. Test for bile salts& bile

pigments:
Test tube (A) Presence of bilesalts
a)Hay's test (for bile salt)
Take two test tubes one with containing sulphur

5ml urine (A) and other with 5ml powder sinks.

water (B). Now gently sprinkle
flowers of sulphur into both.

54 | P age
 b) Fouchet's test (For bile
No Green Colour Absence of bile
pigments)
Take 5ml of urine add a few appea pigrnents

crystals of magnesium sulphate

and shake the tube till it
dissolves. Now add 10% barium
chloride in excess (about 10ml).
A precipitate of barium sulphate
isformed. The bile pigments get
adsorbed to the precipitate of
barium sulphate. Filter the
contents of the tubes, the filter
may be discarded. Dry the
precipitate by using flter paper
To the dry precipitate add a
drop of fouchets reagent that

contains ferric chlorides as the

oxidising agent.

6. Test for Urobilinogen and

porphobilinogen: No Chloroform layer Absence of

Ehrlich's diazo test: changes to pink colour Urobilinogen
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well|
and allow it to stand for 10No Aqueous layer Absence of
mts.Add 5ml of saturated
changes to pink colour Porphobilinogen
sodium acetate and mix. Now

add 5ml of chloroform. Shake

vigorously for a few seconds

and allow the layers to separate.

REPORT:

The given unknown sample contains

55 P a ge
 1. Reducing sugar

2. Bile salts

56|P age
EX.NO: DATE:
URINE
QUALITATIVE ANALYSIs OF ABNORMAL CONSTITUENT OF
SAMPLE- I

OBSERVATION INFERENCE
S.NO. EXPERIMENT
Test For Protein: Presence of protein
Coagulation occurs
a)Heat coagulation test

Fill 4 of a test tube with the

urine acidified with 2% acetic

acid mix & heat the upper

portion.

b) Sulphosalicylic acid test

Presence of protein
To 5ml of urine add 1ml of White precipitate is
20% Sulpho salicylic acid formed

c)Heller's test A white ring of meta | Presence of protein

Take 3ml of con. Nitric acid proteins appears at the

add 2ml of urine along the sides junction of the fluids.

of test tube.

2. Test for blood:

a)Ortho-Tolidine test
Absence of haemoglobin
To 6 drops of freshly prepared
No
transientdark green
0-Tolidine add 6 drops of Colouris formed

hydrogen peroxide and 4 drops

of previously boiled urine.

b)Benzidine test:
Absence of haemoglobin
To 6 drops of freshly prepared
of No transient dark green
benzlidine add 6 drops
colourisformed
hydrogen peroxide and 4 drops
of previously boiled urine.

57 P age
 3. Test for reducing sugar:
Benedict's test: No colour changes from Absence of reducing
blue to orange sugar.
To 5ml of Benedicts
reagent add 8 drops of urine precipitate
sample. Boil over a flame for 2
minutes place
or in a
boiling
water bath for three minutes and
allow to cool.

Test for ketone

bodies: a)Rother's test
Take 5ml of urine and fully
Absence of acetone
saturated with solid ammonium No
permanganat
sulphate. This is to
remove
e colour appears
substances, which may interfere
with the test. Then, add5 drops
of a freshly prepared solution of

sodium nitro prusside and gently

shake. Then add a few ml of

con.ammonia and mix it.

bGerhardt's test
No wine red colour Absence of aceto acetic
To about 5ml urine in a test
appear acid
tube, add drop wise 10% ferric
chloride

5. Test for bile salts & bile

pigments:
a)Hay's test: (for bile salt) Test tube (A) Presence of bile salts

Take two test tubes one with containing sulphur

5ml urine (A) and other with 5ml powders sink.

water (B). Now gently sprinkle

flowers of sulphur into both.

58 P a ge
 b) Fouchet's test (For bilee
No Green colour Absence of bile
pigments)
appea pigments
Take 5ml of urine add a few

crystals of magnesium sulphate

and shake the tube till it
dissolves. Now add 10%
barium chloride in excess
(about 10ml). A precipitate of
barium sulphate is formed. The
bile pigments get adsorbed to
the precipitate of barium
sulphate. Filter the contents of |
the tubes, the filter may be
discarded. Dry the precipitate
by using filter paper. To the dry
precipitate add a drop of
fouchets reagent that contains
ferric chlorides as oxidising the

agent.

6. Test for Urobilinogen and

No Chloroform layer Absence of
porphobilinogen:
Ehrlich's diazo test changes to pink colour Urobilinogen

To 5ml of urine add 5ml of

ehrlichs diazo reagent mix well
and allow it to stand for 10 No Aqueous layer Absence of
mts.Add 5ml of saturated changes to pink colour Porphobilinogen
sodium acetate and mix. Now
add 5ml of chloroform. Shake|
few seconds
vigorously for a

and allow the layers to separate.

REPORT:
unknown sample contains Proteins
The given
Bile salts

59|Page
 She
ENZYMATIC STUDIES
EX.NO:
DATE:
HYDROLYSIS OF STARCH BY SALIVARY AMYLASE(PTYALIN)
AIM
To hydrolyze the starch by using your own salivary amylase enzyme.
PRINCIPLE
reducing sugars.
Alpha-amylase catalyses the hydrolysis of alpha-1,4-linkages of starch and produces
T h e digested starch is removed and treated with dilute iodine at intervals ,unit no blue colouration is produced
The time taken to reach this achromatic point is determined. Sodium chloride acts as an activator ior alpha-

Amylase enzyne.

REQUIREMENTS
Starch Solution (1%).

Sodiurm chloride solution (0.5%).

Buffer Solution (pH 6.8).

lodine Solution (0.01 N).

Diluted Saliva (Clean your mouth water first. Take about 2(0ml of 0.2% sodium chloride in your mouth;|
move up and down with the help of tongue for 2minutes. Collect in glass test tube and shake vigorously. Filten
to remove any epithelial cells present)

PROCEDURE

Measure 5ml of 1% starch solution into a clean test tube.

Add 2ml of buffer solution (pH-6.8) and 2ml of 0.5% sodium chloride to it

The tube is placed in a water bath at 40 0C to warm it.

And in the same water bath 2ml diluted saliva is also kept in another test tube.

A series of test tubes are prepared, each containing 2ml of distilled water and 2 drops of diluted iodine (0.01 N).

Add 2ml of diluted saliva to the wam starch solution. Mix well and note time.

Transfer at intervals 1-2 drops ofdigested mixtures to one of iodine tube.

Shake well and note the colour, which is blue at first

Note the time at which no colour is produced. This is said to be "Chromic period" which is an inverse measur
of the activity of an enzyme.

REPORT

60 |P ag e