PRACTICAL LAB MANUAL

BIOCHEMISTRY & CLINICAL

PATHOLOGY
 INDEX
Expt. Date of Expt.
Name of Experiments Signature
No. done
1. Qualitative analysis of carbohydrates (Glucose).
2. Qualitative analysis of carbohydrates (Fructose).
3. Qualitative analysis of carbohydrates (Lactose).
4. Qualitative analysis of carbohydrates (Sucrose).
5. Qualitative analysis of Proteins (Albumin).
6. Qualitative analysis of Proteins (Casein).
7. Qualitative analysis of amino acids (Tryptophan).
8. Qualitative analysis of amino acids (Tyrosine).
9. Qualitative analysis of lipids (Triglycerides).
10. Qualitative analysis of lipids (Cholesterol).
11. Determination of constituents of urine (glucose).
12. Determination of constituents of urine (Creatinine).
To analyze the given sample of urine for its normal inorganic
13.
constituents.
To analyze the given sample of urine for its normal organic
14.
constituents.
To analyze the given sample of urine for its abnormal
15.
constituents.
To analyze the given sample of urine for its abnormal
16.
constituents.
17. Determination of constituents of blood/serum glucose in
blood
Determination of constituents of blood/serum Cholesterol in
18.
blood.
Determination of constituents of blood/serum
19. glutamateoxaloacetate transaminase (S.G.O.T) in blood
serum.
Determination of constituents of blood/ Serum
20. glutamatepyruvate transaminase (S.G.P.T) in blood serum.
 Experiment: 1 Sl No.: Date:

Aim: Qualitative analysis of carbohydrates (Glucose).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali prakashan;
2020; 29th Edition; page no:5-6

Materials required: Beaker, water bath, test tubes, graduated pipettes, funnel, filter paper, watch
glass, slide, microscope.

Chemical Required: Molisch’s Reagent, Sulfuric Acid, water, Fehling's solution A, Fehling's
solution B, Barfoed’s reagent, Phenyl hydrazine hydrochloride, Acetate buffer, water.

Procedure:

Sl. Test Observation Inference

No
1 Molisch's test: Mix 2ml of Violet/purple ring at the Glucose is present.
Glucose sample with 5 drops of junction of two liquids.
Molisch’s Reagent in a test
tube.Add gently through the side
by tilting the tube, about 2 ml of
concentrated H2SO4 so as to
form a bottom layer.

2 Solubility test: Mix the glucose It is Soluble in water. Indicate the presence of
sample with water properly. glucose.

3 Fehling's test: 2ml of Fehling's Yellow or brick red Reducing sugar present
solution A and 2ml of Fehling's precipitate observed. (Glucose).
solution B is added to the
Glucose solution and boil it in
water bath
 Sl. Test Observation Inference
No
4 Barfoed‘s test: Add 2 ml of Brick red precipitate is Reducing sugar present
Barfoed’ reagent in 2ml solution observed at the bottom of (Glucose).
of glucose sample and keep in test tube.
boiling in water bath and cool for
2 minutes.
5 Osazone test: Set up a boiling Greenish yellow needle Glucosazone i .e. glucose
water bath. Take test tube; add 1 g shaped crystals observe. present
of Phenyl hydrazine
hydrochloride, 2ml of acetate
buffer, pH5.0. Add 5ml of water
mix well and warm gently. Filter
it. To the filtrate add 5 ml glucose
solution and keep in boiling water
bath and cool. Mount the crystals
under microscope and examine
under microscope.

Observation:
Report:

Teacher’s Signature
 Experiment: 2 Sl No.: Date:

Aim: Qualitative analysis of carbohydrates (Fructose).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali prakashan;
2020; 29TH Edition; Page no:5-6

Materials required: Beaker, water bath, test tubes, graduated pipettes, funnel, filter paper, watch glass,
slide, microscope.

Chemical Required: Fructose, Molisch’s Reagent, Sulfuric Acid, Fehling's solution A , Fehling's
solution B, Barfoed’s reagent ,Phenyl hydrazine hydrochloride, Acetate buffer, fougler's reagent, water.

Procedure:

Sl. Test Observation Inference

No
1. Molisch's test: Mix 2ml of Fructose Violet/purple ring at the Fructose is present.
sample with 5 drops of junction of two liquids.
Molisch’s Reagent in a test
tube..Add gently through the side by
tilting the tube, about 2 ml of
concentrated H2SO4 so as to form a
bottom layer.

2. Solubility test: Mix the Fructose It is soluble in water. Indicate the

sample with water properly. presence of
Fructose.

3. Fehling's test: 2ml of Fehling's Yellow or brick red Reducing sugar

solution A and 2ml of Fehling's precipitate observed. present (Fructose).
solution B is added to the Fructose
solution and boil it in water bath.
 Sl. Test Observation Inference
No
4. Barfoed‘s test: Add 2 ml of Barfoed’ Brick red precipitate is Reducing sugar
reagent in 2ml solution of Fructose observed at the bottom of present (Fructose).
sample and keep in boiling in water test tube.
bath and cool for 2 minutes.

5. Osazone test: Set up a boiling water Greenish yellow needle Fructose is present.
bath. Take test tube; add 1 g of Phenyl shaped crystals observe.
hydrazine hydrochloride, 2ml of
acetate buffer, pH5.0. Add 5ml of
water mix well and warm gently. Filter
it. To the filtrate add 5 ml Fructose
solution and keep in boiling water bath
and cool. Mount the crystals under
microscope and examine under
microscope.

6. Fougler's test: To 3 ml fougler's Blue colour develops. Fructose confirmed.

reagent add 0.5 ml of Fructose
solution. Boil for one minute.

Observation:

Report:

Teacher’s Signature
 Experiment: 3 Sl No.: Date:

Aim: Qualitative analysis of carbohydrates (Lactose).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali Prakashan;
2020; 29TH Edition; Page no:5-6

Materials required: Beaker, water bath, test tubes, graduated pipettes, funnel, filter paper, watch
glass, slide, microscope.

Chemical Required: Molisch’s Reagent, Sulfuric Acid, Fehling's solution A , Fehling's

solution B, HNO3, Ammonical silver nitrate , NaOH, Phenyl hydrazine hydrochloride, acetate
buffer, water.

Procedure:

S. Test Observation Inference

No
1 Molisch's test: Mix 2ml of Lactose Violet/purple ring at Lactose is present
sample with 5 drops of the junction of two
Molisch’s Reagent in a test liquids.
tube..Add gently through the side by
tilting the tube, about 2 ml of
concentrated H2SO4 so as to form a
bottom layer.

2 Solubility test: Mix the Lactose sample It is Soluble in water Indicate the presence
with water properly. of Lactose

3 Fehling's test: 2ml of Fehling's Yellow or brick red Reducing sugar

solution A and 2ml of Fehling's precipitate observed present(Lactose)
solution B is added to the Lactose
solution and boil it in water bath .

4 Mucic acid test: To 1 ml Lactose Broken glass like Lactose is present

solution add 1 ml concentrated HNO 3, crystals obtained
boil and cool. which can be
identified under
microscope.
5 Silver Mirror test:1 ml Lactose Silver mirror is Lactose is present
solution add 1 ml Ammonical silver Observed.
nitrate solution add excess NaOH
Warm.
6 Osazone test: Set up a boiling water Badminton ball, Lactosazone i.e.
bath. Take test tube, add 1 g of Phenyl powder puff shaped Lactose present.
hydrazine hydrochloride 2 ml of Crystals.
acetate buffer, PH 5.0. add 5ml of
water mix well and warm gently.
Filter it. To the filtrate add 5 ml sugar
solution and keep in boiling water
bath and cool. Mount the crystals
under microscope and examine under
Microscope.

Observation:

Report:

Teacher’s Signature
 Experiment: 4 Sl No.: Date:
Aim: Qualitative analysis of carbohydrates (Sucrose).
Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali Prakashan;
2020; 29TH Edition; 5-6

Materials required: Beaker, water bath, test tubes, graduated pipettes, funnel, filter paper, watch
glass, slide, microscope.

Chemical Required: Molisch’s Reagent, Sulfuric Acid, wate, Seliwanoffs reagent, alpha- napthol
Solution, HCL, sodium hydroxide solution.

Procedure:

Sl. Test Observation Inference

No.
Molisch's test: Mix 2ml of Sucrose Violet/purple ring at Sucrose is present
sample with 5 drops of the junction of two
Molisch’s Reagent in a test liquids.
1
tube.Add gently through the side by
tilting the tube, about 2 ml of
concentrated H2SO4 so as to form a
bottom layer.

2 Solubility test: Mix the Sucrose It is Soluble in water. Indicate the

sample with water properly. presence of
Sucrose.

3 Seliwanoff’s Test: To 3 ml of Red colour or red Ketoses like

Seliwanoff’s reagent add 1 ml of Precipitate is observed. Sucrose present.
Sucrose solution and heat the mixture
to boil for 2 minutes cool.

4 Rapid furfural test: To 2 ml of Deep purple colour is Ketoses like

Sucrose solution adds 1 ml of alpha- Observed. Sucrose present.
napthol solution (1% in alcohol) and 5
ml concentrated HCL boil.
 5 Inversion test: To 25 ml of Sucrose Red/yellow/green Sucrose is present.
solution add 5 ml of concentrated HCI precipitate is
boil for 3 minutes and cool under tap Observed.
water. Neutralize with sodium
hydroxide solution.

Observation:

Report:

Teacher’s Signature
 Experiment : 5 Sl No.: Date :

Aim: Qualitative analysis of Proteins (Albumin).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; 12-16.

Materials required: Test tubes, Test tube holder, Dropper, Water bath, Stirrer.

Chemical Required: Copper sulphate, Sodium hydroxide, Nitric acid, Millon’s reagent,
Mercuric sulphate, Sodium nitrite, Sulphuric acid, pyridine solution, Ninhydrin reagent,
Distilled water, lead acetate, mercuric nitrate, sulphosalicylic acid, Esbach’s reagent,
saturated ammonium sulphate.

Procedure:

Physical Test:

Sl. No Test Observation Inference

1 Appearance of a solution Turbid solution Albumin, Globulin, Casein

2 Colour of solution Opalescent of milky Albumin, Casein

3 Smell Egg smell Albumin

4 Litmus Test Neutral Albumin, Gelatin

Precipitation test of Proteins

Sl. No. Test Observation Inference

1. Precipitation by heavy metals
2ml of alkaline sample + 2-3 White ppt Protein present
drops of 2% lead acetate
solution.
2ml of alkaline sample + 2 drop Brownish ppt Protein present
of 5% mercuric nitrate solution.
2. Precipitation by alkaloid reagents
 2 ml of sample solution + 3-4 White ppt Protein present
drops of 20% sulphosalicylic
acid.
2 ml of sample solution + 1 ml Yellow ppt Protein present
Esbach’s reagent.

Note-For these test sample solution is made alkaline by using 1% Na2CO3 solution used.

Chemical Test:

Sl. No. Test Observation Inference

1 Biuret Test:
1. Take the given sample to be
tested in a clean test tube. Appearance of bluish Presence of protein.
2. Add 2 ml of sodium violet colour indicates.
hydroxide solution to it.
3. To that add 5 to 6 drops of
copper sulfate solution to it.

2 Xanthoproteic Test:
1. Take 2ml of given Yellow precipitate
sample compound in a indicated. Presence of protein is
test tube. confirmed.
2. Add a few drops of
concentrated sulphuric
acid and heat.
3 Millions Test:
1. Take 2ml of given sample
solution in a clean test Brick red precipitate Presence of protein.
tube. indicates.
2. Add 2-3 drops of Millon’s
reagent and shake well.
3. Observe the change.
4 Ninhydrin Test:
1. Take the sample solution
to be tested in a clean test
tube. Appearance of blue Presence of protein.
2. Add 1-2ml of ninhydrin colouration.
solution to it.
3. Boil the mixture and
observe the change.
Specification test for Albumin

Sl. No. Test Observation Inference

1. Half saturation test: 5ml of given Violet colure observes.
sample with 5ml of saturated
Presence of albumin.
ammonium sulphate solution keep
for 5 min than precipitate obtained
and filter it. Filtreate add with 2ml
of 40% NaOH and 5 drops of 1%
CUSO4 solution.
2. Heller’s test: 2ml of conc. HNO3 A white ring at the
with 2ml of given sample containing junction of two
from the side of the test tube (without Presence of albumin.
fluids observes.
shaking the tube).

Observation:
Report:

Teacher’s Signature
 Experiment: 6 Sl No.: Date:

Aim: Qualitative analysis of Proteins (Casein).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.12-16.

Materials required: Test tubes, Test tube holder, Dropper, Water bath, Stirrer.

Chemical Required: Copper sulphate, Sodium hydroxide, Nitric acid, Millon’s reagent,
Mercuric sulphate, Sodium nitrite, Sulphuric acid, pyridine solution, Ninhydrin reagent,
Distilled water, lead acetate, mercuric nitrate, sulphosalicylic acid, Esbach’s reagent, C.P.R.
Indicator.

Procedure:

Physical test:
Sl. Test Observation Inference
No.

1 Appearance of a solution Turbid solution Casein, Albumin,

Globulin

2 Colour of solution Opalescent of milky Casein, Albumin

3 Smell Milk like smell Casein

4 Litmus Test Alkaline Casein, Metaprotein

Precipitation reactions of Proteins:

Sl.No. Test Observation Inference

1 Precipitation by heavy metals

2ml of alkaline sample + 2-3 drops White ppt Protein present

of 2% lead acetate solution.

2ml of alkaline sample + 2 drops of Brownish ppt Protein present

5% mercuric nitrate solution.

2 Precipitation by alkaloid reagents

 2 ml of sample solution + 3-4 drops White ppt Protein present
of 20% sulphosalicylic acid.

2 ml of sample solution + 1 ml Yellow ppt Protein present

Esbach’s reagent.

Note-For these test sample solution is made alkaline by using 1% Na2CO3 solution used.

Chemical test:

Sl. Test Observation Inference

No.
1 Biuret Test:
1. Take the given sample to be Appearance of bluish Presence of protein.
tested in a clean test tube. violet colour indicates.
2. Add 2 ml of sodium
hydroxide solution to it.
3. To that add 5 to 6 drops of
copper sulphate solution to it.
2 Xanthoproteic Test:
1. Take 2ml of given Yellow precipitate
sample compound in a indicated. Presence of protein is
test tube. confirmed.
2. Add a few drops of
concentrated sulphuric
acid and heat.
3 Millions Test:
1. Take 2ml of given sample
solution in a clean test tube. Brick red precipitate Presence of protein.
2. Add 2-3 drops of Millon’s indicates.
reagent and shake well.
3. Observe the change.

Specific test for casein

Neumann's test

5 ml. of sample solution + 3 Ppt produces. Casein present.

drops of C.P.R. Indicator.
Solution turns pink + 1%
acetic acid drop wise till
colour changes to yellow.
 Above ppt + 3-4 drops of Shinnig yellow or cannary Casein present.
conc. H2SO4 + 10-12 drops yellow colour is obtained.
of conc. HNO3 .Heat until
mixture is colourless.(Add
more HNO3) if
necessary)cool + few ml of
ammonium molybdate
solution.

Observation:

Report:

Teacher’s Signature
 Experiment: 7 Sl No.: Date:

Aim: Qualitative analysis of amino acids (Tryptophan).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.12-16. Materials required: Test tubes, Test tube
holder, Dropper, Water bath, Stirrer.

Chemical Required: Copper sulphate, Sodium hydroxide, Nitric acid, Millon’s reagent, Sodium
nitrite, Sulphuric acid, pyridine solution, Ninhydrin reagent, Distilled water.

Chemical Test:

Sl. No. Test Observation Inference

1 Biuret Test:
1. Take the given sample to be Appearance of bluish Presence of amino
tested in a clean test tube. violet colour indicates. acid
2. Add 2 ml of sodium
hydroxide solution to it.
3. To that add 5 to 6 drops of
copper sulphate solution to it.

2 Xanthoproteic Test:
1. Take 2ml of given Yellow precipitate Presence of amino
sample compound in a indicated. acid is confirmed.
test tube.
2. Add a few drops of
concentrated sulphuric
acid and heat.
3 Aldehyde Test: 2 ml of sample
solution + 5 drops of Million’s Violet ring is formed at Presence of amino
reagent + 5 drops formalin mix + the junction. acid.
2 ml of conc. H2SO4 from the
side of test tube.
 4 Ninhydrin Test:
1. Take the sample solution to Appearance of blue Presence of amino
be tested in a clean test tube. colouration. acid.
2. Add 1-2ml of ninhydrin
solution to it.
3. Boil the mixture and observe
the change.

Observation:

Results:

Teacher’s Signature
 Experiment: 8 Sl No.: Date:

Aim: Qualitative analysis of amino acids (Tyrosine).

Reference: S. R. Kale; R. R. Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29th Edition; Page no.12-16.

Materials required: Test tubes, Test tube holder, Dropper, Water bath, Stirrer.

Chemical Required: Copper sulphate, Sodium hydroxide, Nitric acid, Millon’s reagent, Sodium
nitrite, Sulphuric acid, pyridine solution, Ninhydrin reagent, Distilled water.

Chemical Test:

Sl.No. Test Observation Inference

1 Biuret Test:
1. Take the given sample to be Appearance of bluish Presence of amino
tested in a clean test tube. violet colour indicates. acid
2. Add 2 ml of sodium hydroxide
solution to it.
3. To that add 5 to 6 drops of
copper
sulphate solution to it.
2 Xanthoproteic Test:
1. Take 2ml of given Yellow precipitate Presence of amino
sample compound in a indicated. acid is confirmed.
test tube.
2. Add a few drops of
concentrated sulphuric
acid and heat.
3 Millions Test:
1. Take 2ml of given sample Brick red precipitate Presence of amino
solution in a clean test tube. indicate acid.
2. Add 2-3 drops of Millon’s
reagent and shake well.
Observe the change.

4 Ninhydrin Test:
1. Take the sample solution to be Appearance of blue Presence of amino
tested in a clean test tube. colouration. acid.
2. Add 1-2ml of ninhydrin
solution to it.
3. Boil the mixture and observe the
change.
Observation:

Results:

Teacher’s Signature
 Experiment: 9 Sl No.: Date:

Aim: Qualitative analysis of lipids (Triglycerides).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.17-18.

Materials required: Test tubes, Test tube holder, Water bath, Stirrer, hot plate.

Chemical Required: Water, Alcohole, NaOH, Nacl, Soap, Cacl2, HCL, Triglyceride.

Procedure:

Physical test:

Fats are colourless, odourless and tasteless in chemically pure form. But in natural or rancid
state they are associated with same colour or aroma.

Solubility test:

Sl. Test Observation Inference

No.
1 1)Triglyceride + water – Warm Immiscible separate Triglycerides are
gently layers are formed. insoluble in water.
2) Triglycerides + Organic solvents soluble Triglycerides are
like chloroform, alcohol, ether etc. soluble in organic
solvents.
2 Litmus test No change in colour or Triglycerides are
Test the solution of Triglycerides red litmus paper. neutral in nature
with blue or red litmus.
3 Specific gravity Triglycerides are float Its specific gravity is
Add small quantity of Triglycerides on water less than one.
to a test tube full of water.
Chemical test:

S.No Test Observation Inference

1 Emulsification: A white homogenous Triglycerides form

emulsion is formed emulsion in water,
1) 1 ml alcoholic which breaks on which breaks on
solution of Triglycerides + standing by separating
5ml of Standing.
fatty phase in the form
Distilled water shakes vigorously. of oily droplets.

A white Triglycerides form

2) 1 ml. alcoholic
homogenous emulsion in water
solution of Triglycerides + emulsion is formed which can be
which remains stable stabilised by
5ml. of 1 % bile salt solution on standing emulsifying agent like
+ 5ml. distil bile salt.

Water, shake vigorously.

 2 Saponification: 10 ml. of 20% NaOH
+ alcoholic solution of Triglycerides
10ml. Heat on water both, until a drop
of this mixture do not separate oil
drop when added

To distil water. Add the mixture

with equal quantities of distil water
and divide in Three equal parts.

a) 2 mL of above mixture + Cake of soap floats on Triglycerides is

top separate this cake saponified in to
1) A large knife point of solid Nacl, and makes two parts.
shake vigorously.

Nothing takes place. Formation of soap

2) One part of above soap + 10, 20 ml
water shake. conformed.

ppt dissolves. Sodium soap is soluble

in water.
3) Second part of soap+ 5ml distils
water.
Calcium soap is
A white flocculant ppt insoluble in water.
b) 2 ml of above mixture + 3ml of 2% is formed insoluble in
Cacl2 solution shake. water

c) Above mixture 3ml + 2/3 drops of Triglycerides separates Soap salt of fatty acids
conc. Sulphuric or hydrochloride and floats on water is converted into fatty
acid boil. acids on treatment
with HCL/H2SO4

Observation

Results:

Teacher’s Signature
 Experiment: 10 Sl No.: Date:

Aim: Qualitative analysis of lipids (Cholesterol).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.19.

Requirement:
Materials required: Test tubes, Test tube holder, Stirrer, Microscope.

Chemical Required: Water, chloroform, H2SO4, acetic anhydride. Procedure:

Physical test: It forms white shining rhombic crystals having notched corners.

Chemical test:

Sl.No. Test Observation Inference

1 a) Solubility

i) Cholesterol + water Insoluble. Being lipid it is

ii) Cholesterol + organic solvent. insoluble in
Soluble. water. Being lipid
it is soluble in
b) Microscopic appearance organic solvent.
White shining rhombic
crystal.
2 c) Salkiwaski’s test: Upper chloroform Presence of
layer shows red cholesterol is
2 ml cholesterol solution in coloration while confirmed.
chloroform + slowly add 2 ml conc. lower H2SO4 layer
H2SO4 wait for 3 min. shows green
Fluorescence.
3 d)Libermann-Burchrdt’s test: A rose red colour Presence of
develops which cholesterol is
2ml of cholesterol solution in quickly changes to confirmed.
chloroform + 10 drops of acetic blue to green.
anhydride + 2 drops of conc. H2SO4.

Observation:
Results:
Teacher’s Signature
 Experiment: 11 Sl No.: Date:

AIM: Determination of constituents of urine (glucose).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition

Requirement:
Material requirements: Flask (50ml), Pipette (1-10ml), Photoelectric Colorimeter.
Chemicals requirements:
Principle:
The urine glucose test involves taking a sample of urine. Once you provide your sample, a
small cardboard device known as a dipstick will measure your glucose levels. The dipstick
will change color depending on the amount of glucose in your urine.
A urine glucose test is a quick and simple way to check for abnormally high levels of glucose
in your urine. Glucose is a type of sugar that your body requires and uses for energy. Your
body converts the carbohydrates you eat into glucose.
Having too much glucose in your body can be a sign of a health problem. If you don’t receive
treatment and your glucose levels remain high, you can develop serious complications. The
urine glucose test involves taking a sample of urine. Once you provide your sample, a small
cardboard device known as a dipstick will measure your glucose levels.
The dipstick will change color depending on the amount of glucose in your urine. If you have
a moderate or high amount of glucose in your urine, your doctor will perform further testing
to determine the underlying cause.
The most common cause of elevated glucose levels is diabetes, a condition that affects your
body’s ability to manage glucose levels. It’s important to monitor your glucose levels if you
have already been diagnosed with diabetes, or if you show symptoms of prediabetes. These
symptoms include:
• excessive thirst
• blurred vision fatigue

Procedure:

Benedict's Test

Materials required:
Test tube, test tube holder, urine sample, measuring cylinders, Benedict’s solution and burner.
Procedure:

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample in it.
• Take 5 ml Benedict’s reagent in a measuring cylinder.
• Add Benedict’s reagent to the test tube that contains urine sample.
• Using a test tube holder, hold the test tube firmly and heat it for 2 minutes on the burner.
• Keep shaking the test tube while heating.
• A yellow precipitate appears which indicates the presence of sugar in urine.
• Depending upon the concentration of sugar in the urine, either green, yellow, or brick red
precipitates are formed.
Fehling's test

Materials required
Test tube, test tube holder, urine sample, measuring cylinders, Fehling’s solution A, Fehling’s
solution B and burner.

Procedure

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample in it.
• Take 2 ml Fehling’s solution A in a measuring cylinder.
• Add Fehling’s solution A to the test tube that contains urine sample.
• Take 2 ml Fehling’s solution B in a measuring cylinder.
• Add Fehling’s solution B to the test tube that contains urine sample.
• Using a test tube holder, hold the test tube firmly and heat it gently for 2 minutes on the
burner.

• Keep shaking the test tube while heating.

• A green precipitate appears which indicates the presence of traces of sugar in urine.
• Depending upon the concentration of sugar in the urine, either green, yellow or brick red
precipitates are formed. Simulator Procedure (as performed through the Online Labs)You
can select the test from the ‘Select type of test’ drop down list.

Benedict's Test
• Drag the dropper containing Benedict’s reagent towards the test tube to pour the reagent
into it.

• Click on the knob of the burner to turn it on.

• Drag the test tube towards the burner to heat it.
• Click on the information icon to see the inference.
• You can redo the experiment by clicking on the ‘Reset’ button.

Fehling’s Test

• Drag the dropper containing the Fehling’s reagent A towards the test tube to pour the

reagent into it.

• Drag the dropper containing the Fehling’s reagent B towards the test tube to pour the

reagent into it.

• Click on the knob of the burner to turn it on.

• Drag the test tube towards the burner to heat it.
• Click on the information icon to see the inference.
• You can redo the experiment by clicking on the ‘Reset’ button.

Observation:
Report:

Teacher’s Signature
 Experiment: 12 Sl No.: Date:

Aim: Determination of constituents of urine (Creatinine).

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.38-39. Requirement:

Material requirements: Flask (50ml), Pipette (1-10ml), Photoelectric Colorimeter.

Chemicals requirements: Picric acid (1%), NaOH (10%), Standard creatinine solution.

Principle:

Creatine in urine is estimated by Polin modified method using "Photoelectric colorimeter". In

this method creatinine present in the given sample of urine is estimated by Folin modified
method. In this method, urine sample containing Creatinine is treated with picric acid in
alkaline medium to obtain red coloured creatining picrate. Optical density of this red
coloured solution is compared with that of standard solution similarly converted by picric
acid to creatinine picrate. By using calorimetry principle concentration of creatinine in given
sample of urine can be calculated.

In second part of the experiment urine sample is boiled with acid so that creatine present in
urine gets converted into creatinine. Then total creatinine (creatinine present in urine +
creatinine obtained by conversion of creatine) in urine is estimated by the same method.
So

Creatine obtained by conversion of creatine = Total creatinine - Creatinine present in the

Creatine present in the sample Creatinine obtained by Conversion of creatine

Conversion factore of creatine to creatinine.

Creatinine obtained by Conversion

of creatine 1.16 as

1mgof creatinine=1.16mg of creatine.

Procedure:
Step 1. Estimate the mg. % of creatinine in given sample of urine as explained in previous
Experiment (suppose 'A' mg.%).
Step 2. In a 250 ml. flask pipette 0.5 ml of urine and l0 ml of picric acid. Add few porcelain
Pieces. Add 150 ml of water. Boil gently for 45 minutes and then rapidly till the volume is
Reduced to about 10 ml. In this procedure creatine present in the urine sample is converted
Into creatinine. Now estimate the mg % of total creatinine (creatinine present + creatinine
Obtained from creatine) by similar method explained in previous method. (Suppose 'B' mg.
%).

Calculation

Report

Patient’s name:

Sample number:

Urine creatine

value: (Estimated)
Teacher’s Signature
Urine creatine

value: (Normal)

Date:

Sign:
 Experiment: 13 Sl No.: Date:

AIM: To analyze the given sample of urine for its normal inorganic constituents.

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali

Prakashan; 2020; 29TH Edition; Page no.19.

Principle:
The principle inorganic constituents of urine are chlorides, phosphates, sulfates and ammonia.
Sodium chloride is the predominant chloride and makes up about half of the inorganic
substances.
Requirements
Sample: Human urine sample
Apparatus: Test strips, Beaker
Chemicals: Then add distilled water to make the total volume 1000 ml., Nitric acid solution –
2, Silver nitrate solution – 2N, sodium molybdate, dil. Hcl, of 5% barium chloride solution,
Sodium hydroxide solution Procedure:

S. Chemical Test Observation Inference

No.

1. Chlorides To about 5 ml of Formation of white Indicates the presence

urine sample add 5 precipitate of chloride
drops of 2N nitric
acid and 2N silver
nitrate solution.
2. Phosphates To about 5 ml of Appearance of yellow Indicates the presence
urine sample add crystalline precipitate of phosphate in urine.
nitric acid because of ammonium
5 ml and sodium phosphor-molybdate
molybdate 4 ml and
heat it for about 5
minutes.
3. Sulfates At first take 10ml of A white colour Indicates presence of
urine and to it add 1ml precipitate is sulphate.
dil. Hcl and then add 4 formed because of
drops of 5% barium barium sulfate
chloride solution.
 4. Ammonia Take 5 ml of urine and Now indicate the Indicates presence of
to it add 10% NaOH presence of evoluted ammonia
solution of volume ammonia by the help
1ml. of red litmus paper.

Observation:
Report:

Teacher’s Signature
 Experiment: 14 Sl No.: Date:

AIM: To analyze the given sample of urine for its normal organic constituents.
Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.19.

Requirements:
Sample: Human urine sample
Apparatus: Test strips, Beaker
Chemicals: Mercuric nitrate solution, Oxime reagent, picric acid solution, Benedict’s reagent

Principle:
Urine is the waste product formed by the kidney that is excreted by body and helps in
maintaining the electrolyte and pH level of the body. 1 Urine is composed of various organic
and inorganic constituents. So the basic objective of this experiment is to study about various
organic and inorganic constituents of urine through various tests.

Procedure:
Sl. Chemical Test Observation Inference
no.

1. Test for Urea

a) Mercuric
nitrate solution

b) Oxime
reagent

2. Creatinine ( Take about 5 ml of When the solution It indicates

Alkaline urine sample and add becomes alkaline it the presence
picrate test) 1 or 2 drops of changes to red colour the creatinine
saturated picric acid or orange colour
solution. Then add because of formation
sodium hydroxide of creatinine picrate
solution.
 3. Uric acid Take urine sample 2 If a white precipitate It indicates the
ml and to it add 1 ml is formed it presence of uric acid.
a) Schiff’s of Benedict’s reagent
test and then for about
three minutes heat it
b) Phosphate in hot boiling water
tungstate bath
reagent test

Observation:
Report:

Teacher’s Signature
 Experiment: 15 Sl No.: Date:

Aim: To analyze the given sample of urine for its abnormal constituents.

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali

Prakashan; 2020; 29TH Edition; Page no.29-31

Requirement:
Material requirements: Flask (100ml), Burette (50ml), Pipette (10ml), Pieces of porcelain.

Chemicals requirements: Benedict’s quantitive reagent, Anhydrous/Crystalline sodium

carbonate, Distilled water.

Principle:

Benedict's solution (quantitive), which is copper sulphate in alkaline solution, is reduced by

glucose. The benedict's quantitative reagent consists of copper sulphate, potassium thiocynate
and other chemicals in alkaline media.

Copper sulphate is reduced to cuprous oxide by glucose. The potassium thiocynate reacts with
cuprous oxide and forms a white ppt of cuprous thiocynate instead of usual red precipitate of
cuprous oxide. The disappearance of blue colour /tint from solution indicates complete
reduction of copper sulphate.

2CU (OH)2 CU2O + 2H2O

Procedure:

Benedict's method

 Wash and clean the required apparatus.

 Pipet 10 ml of the benedict’s quantitive reagent in 100ml flask with long narrow neck (So
as to prevent oxidation, by atmospheric oxygen.
 Add 20 ml of water and 5gm of anhydrous sodium carbonate and few pieces of porcelain
 Heat the flask on a flame. It should be noted that the titration mixture must be kept boiling
throughout the titration period.
 Fill the burette with urine (diluted). Adjust the meniscus.
 When the contents in the flasks begin to boil add urine ml wise rapidly until white
precipitate appears.
 After this add urine from burette drop by drop at intervals of 10 seconds.
 Continue the addition of urine from burette until the last trace of blue colour (due to
CUS04) disappears. End point:
 Allow the titration mixture to cool.
 The white ppt settles down.
 Complete disappearance of the blue colour of the benedict's reagent.  The fluid in the
titration mixture must show light green colour on cooling.

Calculation:

Reports:

Patient’s name:

Sample number:

Urine glucose: (Estimated value) Urine glucose: (Normal Teacher’s Signature

value) Date:

Sign:
 Experiment: 16 Sl No.: Date:

Aim: To analyze the given sample of urine for its abnormal constituents.

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali

Prakashan; 2020; 29TH Edition; Page no.36-37. Requirement:

Material requirements: Flask (50ml), Pipette (1-10ml), Photoelectric Colorimeter.

Chemicals requirements: Picric acid (1%), NaOH (10%), Standard creatinine solution.

Principle:

Creatinine in urine is estimated by Folin modified method using photoelectric colorimeter. In

this method, urine sample containing Creatinine is treated with picric acid in alkaline medium
to obtain red coloured creatinine picrate. Optical density of this red coloured solution is
compared with that of standard solution similarly converted by picric acid to creatinine
picrate. By using colorimetry principle concentration of creatinine in given sample of urine
can be calculated. Procedure:

Step 1. Label two flasks of 5O ml capacity as 'S' standard and 'U' unknown.
Step 2. Preparation of standard:
In standard flask (S) add followings:

 Standard creatinine solution - 0.5 ml

 Sodium hydroxide solution - 1.0 ml (NaOH 10%)
 Picric acid (1%)- 10.0 ml. Dilute by adding distil water to 5O ml. Mix and keep for 15
minutes.
Step 3. Preparation of unknown:
In unknown flask (U) add followings:

 Given urine sample- 0.5 ml.

 Sodium hydroxide solution - 1.0 ml. (NaOH 10%)
 Picric acid (1 %)- 10.0 ml. Dilute by adding distil water to make final volume 50 ml. Mix and
keep for 15 minutes
.·
Step4. Record the colours (i.e. optical density) obtained of that of standard and
unknown by using photoelectric colorimeter.
Calculation:

Reports:

Patient’s
name:

Sample
number:
Teacher’s Signature
Urine creatinine

value: (Estimated)

Urine creatinine

value: (Normal)
 Experiment: 17 Sl No.: Date:

Aim: Determination of constituents of blood/serum glucose in blood

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; NIRALI

PRAKASHAN; 2020; 29TH Edition; Page no.41-44. Requirement:

Material requirements: Folins sugar tube, Pipette graduated, flasks, photoelectric colorimeter

Chemicals requirements: Alkaline copper sulphate solution, Phosphomolybdic acid, Sodium

tungstate 10% , Sulphuric acid 2/3 N, Benzoic acid solution, Stock glucose solution, Standard
glucose solution No. 1, Standard glucose solution No. 2, Fluoride oxalate solution.

Principle:
In this method protein free filtrate is obtained (Folin-wu filtrate) so that 10 ml of filtrate
corresponds to 1 ml of blood sample. Protein free filtrate is obtained by precipitating the
proteins of blood by tungstic acid. Then this protein free filtrate containing glucose is heated
with alkaline copper sulphate solution. Thus glucose reduces copper sulphate to form
equivalent quantity of cuprous oxide.

This cuprous oxide formed is reduced with phosphomolybdic acid to produce corresponding
equivalent quantity of molybdenum blue. The molybdenum blue gives intense blue colour,
the "intensity of which is directly proportional to cuprous oxide which corresponds to the
amount of glucose present in given sample of ‘folin-wu" filtrate.

Reaction:

C6H12O6 + CU++ CU+ + Oxidation

Glucose cupric

CU+Na2PO4: 12MoO3 CU++ CU++ 12MoO

Cuprous
The blue colour obtained with test blood sample is compared with standard solution by
similar procedure and by using photoelectric calorimeter. The optical density of test and
standard is measured and concentration of glucose in blood can be calculated using
colorimetric principle. Procedure:
Folin- Wu (modified)
 Wash clean, label three folin-wu tubes as:
• unknown ... 'U
• Standard I - Std I
• Standard II - Std II
 To the folin wu tube labelled as "U" take 2 ml of "Folin wu filtrate".
 In a folin-wu tube labelled as "Std I" take 1 ml of standard sugar solution I (0.1 mg sugar).
4. In a folin-wu tube labelled as "Std II" take 1 ml of standard sugar solutjon Il (0.2 mg
sugar).
 To all above tubes add 1 ml of alkaline copper sulphate solution.
 Keep the tubes in boiling water bath for 6 to 8 minutes.
 Remove from the water bath and add 1 ml of phosphomolybdic acid to all tubes.
 Keep the tubes again in boiling water bath for 2 minutes and after 2 minutes cool to room
temperature.
 Add 25 ml of distil water to each tube mix well and record. Compare the optical densities
by using photoelectric colorimeter by using tube filter 420 mµ.

Calculation:

Reports:

Patient’s

name: Sample

number:

Blood sugar:
Teacher’s Signature
(Estimated)

Blood sugar:

(Normal)

Date:
 Experiment: 18 Sl No.: Date:

Aim: Determination of constituents of blood/serum Cholesterol in blood.

Reference:S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; Nirali
Prakashan; 2020; 29TH Edition; Page no.55-57.

Requirement:
Material requirements: Centrifuge, Test tube, Graduate Pipettes, Photoelectric colorimeter.

Chemicals requirements: Standard cholesterol solution colour reagent.

Method used: Direct method of ferro and ham. Principle:

This is a direct method used for estimation of cholesterol in serum by ferro and hom. In this
method is a mixture of acetic anhydride; glacial acetic acid, and sulphuric acid in appropriate
proportion is used. The colour reagent gives bluish colour with cholesterol. In this method the
colour is developed directly without the extraction of lipids.

In the "standard" preparation two drops of distil water addition is advised, as it hastens the
reaction and develops color. This standard" color with which "unknown" color is compared
by using photo-electric calorimeter.

Procedure:

Preparation of unknown sample:

i. In a test h1be labelled as "U" pipette out 0.2 ml of serum.

ii. Add 5 ml freshly prepared color reagent. iii. Mix well by
shaking and keep the tube in dark for 10 minutes.
iv. Obtain a optical density for unknown by using photoelectric calorimeter at 660 mµ. Record
and note it as "Eu".

Preparation of standard:

 In a tube labelled as 'S' take 0.2 ml of standard cholesterol solution.

 Add 2 drops of distil water and 5 ml of color reagent.
 Mix well by shaking and keep the tube in dark for 10 minutes.
 Obtain an optical density for standard by using photoelectric calorimeter at 660 mu.
Calculation:

Reports:

Patient’s name:

Sample number:

Blood

cholesterol:

(Estimated)

Blood Teacher’s Signature

cholesterol:
 Experiment: 19 Sl No.: Date:

Aim: To estimation of Serum glutamate-oxaloacetate transaminase (S.G.O.T) in blood serum.

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; NIRALI
PRAKASHAN; 2020; 29TH Edition; Page no.66-68.

Requirement:
Material requirements: Centrifuge, Test tube, Graduate Pipettes, Photoelectric colorimeter.
Chemicals requirements: Phosphate (pH 7.4), Stock pyruvate solution, Standard solution,
DNPH solution, S.G.O.T. substrate, Sodium hydroxide.

Principle:

Transaminases are the enzymes which promotes the process of removal of α-amino groups of
most of L amino acids to a α-keto acid. As a result number of alpha amino acids and alpha
keto acids are formed.
One of these are serum asparate transminase i.e. which catalyses the reaction of glutamate
oxalo acetate transaminase i.e.' G.O.T.

L - alpha - oxaglutrate + L asparare L - glutamate + L –oxaloacetate.

This oxaloacetate formed in the reaction with glutamate oxalo acetate transaminase (GOT)
decarboxylates spontaneously to pyruvate which is again measured by hydrazone formation.
The colour obtained is measured in colorimeter at 510 mµ.(filter).

Procedure: [A] Preparation of

unknown sample:

 In a tube labelled as "U" take asparate substrate 0.5 ml.

 Add 0.1 ml serum sample.
 Incubate the tube at 37° c for 30 minutes.
 Remove the tube and add 0. 5ml DNPH solution keep 20 minutes at room temperature.
 Add 5ml of 0.4N NaOH in the tube.
 Obtain the optical density for unknown by comparing the colours by using
photoelectric colorimeter with green filter (520 mµ).

Note it as "Eu".
Preparation of control:

 In a tube labelled as "C" take · 0.5 ml of asparate substrate, 0.5 ml DNPH solution and
0.1 ml serum.
 Incubate the tube at 37° c for 30 minutes.
 After 30 minutes, remove from water bath and keep 20 minutes at room temperature.
  Add 5 ml of 0.4N NaOH to the tube compare the colour by using green filter.

Preparation of standard:

 In a tube labelled as "S" take 0.5 ml of asparate substrate and 0.5 ml of DNPH
solution. Add 0.1 ml of standard-pyruvate.
 Incubate the tube at 37° C for 30 minutes.
 Remove after 30 minutes and keep at room temperature for 20 minutes.
 Add 5 ml of 0.4N NaoH solution.
 Compare the colour by using green filter. Note it as "Es". Preparation of blank

sample:

• In a tube labelled as "B" take:

• 0.5 ml asparate substrate.
• 0.5 ml DNPH solution.
• 0.1ml distils water. Incubate at 37°C for 30 minutes.
• After 30 minutes keep at room temperature for 20 minutes.
• Add 5 ml NaoH (.4N) solution.
• Compare the colour using green filter.
• Prepare unknown control standard, blank as before [SGPT experiment} .
• Incubate tube for 60 minutes at 37° c.
Calculation:
Reports:
Patient’s name:
Sample number:
SGOT:
(Estimated) SGOT: (Normal)
Date: Sign:

Teacher’s Signature
 Experiment: 20 Sl No.: Date:

Aim: To estimation of Serum glutamate-pyruvate transaminase (S.G.P.T) in blood serum.

Reference: S.R.Kale; R.R.Kale: Practical Biochemistry & Clinical Pathology; NIRALI
PRAKASHAN; 2020; 29TH Edition; Page no.64-66.

Requirement:
Material requirements: Centrifuge, Test tube, Graduate Pipettes, Photoelectric colorimeter.
Chemicals requirements: Phosphate (pH 7.4), Stock pyruvate solution, Standard solution,
DNPH solution, S.G.P.T. substrate, Sodium hydroxide.

Principle:

Transaminases are the enzymes which promotes the process of removal of α-amino groups of
most of L-amino acids to α-keto acid. As a result number of alpha amino acids and alpha keto
acids are formed.

One of these are serum alanine-transminase (S.G.P.T.) this catalyses the reaction as follows.

L-alpha oxoglutrate + L alanine L-glutamate + L-pyruvate.

This pyruvate produced by "glutamate-pyruvate-transminase" reacts with di-Nitrophenyl

hydralazine (DNPH solution) in an alkaline medium which is measured at 510 mµ filter.

Note: In the estimation, the concentration of substrate is suboptimal, to reduce background

colour produced by ketoglutarate in the reaction with Di-nitro-phenyl-hydrazine (DNPH).

Procedure:

Preparation of unknown sample:

 In a tube labelled as "U" take alanine substrate 0.5 ml.

 Add 0.1 ml serum sample.
 Incubate the tube at 37° C for 30 minutes.
 Remove the tube and add 0. 5ml DNPH solution keep 20 minutes at room temperature.
 Add 5ml of 0.4N NaOH in the tube.
 ·Obtain the optical density for unknown by comparing the colours by using photoelectric
colorimeter with green filter (520 mµ).

Note it as "Eu".

[B] Preparation of control:

  In a tube labelled as "C' take 0.5 ml of alanine substrate, 0.5 ml DNPH solution and 0.1
ml serum. 2. Incubate the tube at 37°·c for 30 minutes.
 After 30 minutes; remove from water bath and keep 20 minutes at room temperature.
 Add 5 ml of 0.4 N NaOH to the tube compare the colour by using green filters. Note it
as "Ec".

[C] Preparation of standard:

 In a tube labelled as "S" take 0.5 ml of alanine. Substrate and 0.5 ml of DNPH solution.
Add 0.1 ml of standard pyruvate.
 Incubate the tube at 37°C for 30 minutes.
 Remove after 30 minutes and keep at room temperature for 20 minutes.
 Add 5 ml of 0.4 N NaoH solutions.
 Compare the colour by using green filter. Not it as "Es".

[D] Preparation of blank sample:

1. In a tube labelled as "B” takes:

• 0.5 ml alanine substrate

• 0.5 ml DNPH solution
• 0.1 ml distil water
Incubate at 37° c for 30 minutes
2. After 30 minutes keep at room temperature for 20 minutes.
3. Add 5 ml NaoH (.4N) solution.
4. Compare the colour using green filter. Note it as "EB"
Calculation:

Reports:

Patient’s name:

Sample number:

SGPT: (Estimated) SGPT: (Normal)

Date: Sign:
Teacher’s Signature