OMC .

ochemistry

Dhera
of K-7
tment Y

Laboratory
MEDICAL Handbook
Department of
HG
-ES

Biochemistry
ESTD Dhaka Medical College
Batch: K-79
Roll No. :

500m.
AMPROK

-200 200

150
 Department of Biochemistry
Dhaka Medical College, Dhaka

Particulars of the Students (Batch K-79)

Fix up a recent
Name of the Student
photograph

Class Roll No.

Session :

Student's Contact No. :

Student's e-mail Address

Present Address :

Permanent Address

Name of Guardian Contact No. e-mail Address

Father

Mother

Local

GPA Year Institution & Board

Academic SSC/Equivalent
Records

HSC/Equivalent

2
 Laboratory Etiquettes

There are certain rules and regulations, etiquettes and manners

which are supposed to be maintained by everyone in the
laboratory during working and learning.

These are as follows

1. Students should be punctual and regular in there practical

classes so that they can listen and clearly understand.

2. They should be sincere and careful to their practical classes to

avoid exposure to any type of lab hazards.

3. Students must have clear theoretical knowledge about the

experiment.

4. They must wear proper lab attire such as apron, mask, glass,
gloves, shoes, and cap in proper way. Loose dress and half shoes
must be avoided.

5. Each student should have either a timer or a watch.

6. Laboratory environment should be quiet so that all sorts of

noise and confusions can be avoided.

7. When working in a group, everyone should participate equally

in their experiments to gain proper practical knowledge.

8. They should never use unclean pipettes to avoid the risk of

infection.

9. Separate pipette should be used for each reagent.

10. Clean and dry test tubes should be used otherwise result of
the experiment will be hampered.

11. Students should never remove or displace any reagent from

one table to another.

12. Micropipettes should be used in cases of measuring

poisonous or radioactive substances.

3
13. Students should not keep an open inflammable substance
near burner.

14. Eating, drinking etc should be strictly prohibited in the

laboratory,

15. Required apparatus and reagents should be kept in a

protective place.

16. Reagent bottles must not be kept open & don't remove its
leveling sticker.

17. In cases of confusions students should consult with teacher.

18. Before leaving the laboratory students should handover the

apparatus to the concerned person of the laboratory.

19. During preparation of solutions, water should not be poured

into acid.

20. Students should take care of the laboratory.

21. Students should maintain a notebook and bring it to the

laboratory every day.

22. Hands should be kept away from the mouth during working
in the laboratory.

23. Frequent hand washing will be encouraged in the laboratory.

Students should be careful enough about their safety as

well as obeying the manners and etiquettes.
Laboratory Safety & Laboratory
Hazards

Laboratory Safety:
Every clinical laboratory should have a formal safety
program, ensuring that the laboratory environment meets some
accepted safety standards.

This effort includes proper labeling of chemicals, types and

locations of fire extinguishers, proper grounding of electrical
equipments and provisions of means for proper handling and
disposals of bio-hazardous materials including specimens
collected from patients.

Mandated Plans for Laboratory Safety:

There are two different types of mandated plans for
laboratory safety:

A. Chemical Hygiene Plan

B. Exposure control plan

A. Chemical Hygiene Plan:

The suggested elements of chemical hygiene plan are
(a) Glossary of terms
(b) Standard operating procedures
(c) Criteria used to determine and implement the control
measure

(d) Chemical inventory

(e) Environmental monitoring and maintenance
(f) Chemical labeling and storage

B. Exposure control plan:

Biochemical laboratory should develop, implement and

adhere to a plan that ensures a protection of laboratory learners
against potential exposure to blood borne pathogens. It also

5
ensures that, the medical wastes that the laboratory produces
are managed and handled in a safe and effective manner.

The suggested elements of exposure control plan are

a) Wear professional lab attire
(a) To protect exposure of oneself from blood borne
pathogens such as during pricking (apron, plastic disposable
gloves should be used)
(b) To prevent the spread of blood borne pathogens proper
centrifugation & proper disposal of blood borne sample is needed

Laboratory Hazards
Various types of hazards are encountered during the operation of
a clinical laboratory. These hazards must be identified and work
practices must be developed to deal with them. These hazards
include

(a) Biological hazards: from biological samples like blood,

sputum & any other biological fluid.
(b) Chemical hazards: from corrosive chemicals such as
liquids, radioactive chemicals etc.

(c) Electrical hazards: from improper grounding, damaged

extension cords, poor electrical wear, faulty electrical equipment's
etc.

d) Glassware hazards: from broken instruments.

e) Fire hazards: from flammable liquid or solids.

To avoid these types of hazards the following precautions should

be abide by

(a) Avoid mouth pipetting and never blowout pipettes containing

potentially infectious materials.

(b) Do not mix potentially infectious materials by bubbling air

through any liquid.

6
 (c) Use barrier protections such as gloves, musk etc.

(d) Wash hands whenever gloves are changed.

(e) Try to prevent accidental injuries.

(f) Make a habit of keeping your hands away from your mouth

g) Follow proper waste disposal system:

a) Red colored bean: for sharp wastes like needles, glass

syringe, broken glass ware etc.
b) Green colored bean: for recycled material plastic syringe,
saline bag, water bottle etc.
c) Black colored bottle for food peal, blister pack of medicine,
:

waste food etc.

d) Yellow colored bean: for infectious waste like left biological

sample, used cotton, bandages, gauze etc.

7
Introduction to Common Laboratory Glassware and
Apparatus

Name of common glassware and apparatus used in Biochemistry

laboratory:

In the laboratory different types of glassware and apparatus are

commonly used. These are -
1. Pipette
2. Micropipette
3. Graduated measuring cylinder
4. Different types of flasks
5. Funnel

6. Test tube

7. Test tube holder

8. Test tube rack

9. Burner

10. Beaker

11. Disposable syringe

12. Stirrer

13. Tourniquet

Precautions to be taken before using any glassware:

1. Glassware and apparatus should always be free from greasy or

oily substances in order to prevent formation of droplets.

2. Reading should be taken at the level of the meniscus. In cases

of diluted or transparent solutions, reading should be taken at

the lower level of the meniscus. And in the cases of deeply colored
or highly concentrated solutions, reading should be taken at the
upper level of the meniscus.

3. The temperature of the solution, which is to be measured

should be at or near the calibration temperature of the

apparatus.

8
 Uses of glassware and apparatus:

1. Pipette: Two different types of pipettes are seen at the

laboratory
glass
(a) Graduated pipette: Uses
(i) Measurement of any small volume of liquid where higherdegree
of accuracy is required.
(ii) Transfer of liquid from one container to another.

(b) Volumetric pipette- Uses

(i) Measurement of fixed volume of liquid where higher degree of
accuracy is required.
(ii) Transfer of liquid from one container to another.

2. Micropipette: To measure very small amount of liquid sample

accurately.

3. Graduated Measuring Cylinder: Uses

(a) Measurement of large volume of liquid where higher degree of
accuracy is not required.
(b) Preparation of solutions e.g. Normal Saline.

4. Different types of Flasks: uses

(a) Volumetric flask: Used for preparation of solution and
preservation of solution.
(b) Conical flask: Used in titration purposes and heating
purposes.

(c) Flat bottomed flask: Used for indirect heating.

(d) Round bottomed flask: Used for direct heating.

5. Funnel: Uses

(a) Pouring liquid substances into a narrow mouthed container.

(b) Supporting filter paper during filtration.

9
6. Test tube: Uses

(a) To do test in it.

(b) To apply heat in a solution.

(c) Preservation of specimen like blood, CSF, urine etc.

7. Test tube holder: Uses-Holding test tube during heating.

8. Test tube rack: To hold test tubes.

9. Burner: To apply heat in a solution and any type of heating

purposes.

10. Beaker: Uses- (a) Holding solution in the laboratory.

(b) Preparation of solution
(c) As a discarder.

11. Disposable syringe: Use- Collection of blood from vein.

12. Stirrer: Use- To stir during preparation of solution.

13. Tourniquet: Use- Collection of blood from vein.

10
 Volumetric Pipette
Graduated

Pipette

125

1576

Graduated

Measuring
Cylinder
Micropipette 30

11
 Flat

Bottomed

Flask

Volumetric

Flask

-250ml

Round

Bottomed
Flask

LABOY
Conical 1000

Flask
800

1000ml
600

400

12
 Funnel

Test Tube

Test Tube

Holder

Test Tube

Rack

Burner 500
APARICI

400

300
500ml
200
Beaker

100

13
Disposable Tourniquet
Syringe

My

777
Y

Stirrer

14
Preparation of solutions

(a) Preparation of 100ml normal saline from sodium chloride

crystal.

Definition of normal saline: Normal saline is the saline in which

sodium chloride has the same osmotic pressure as in

intracellular fluid of RBC.

Content: 0.9 gm sodium chloride in 100ml solution.

Apparatus & Requirements: (i) Analytical balance (ii) 100ml

volumetric flask or 100ml graduated measuring cylinder
(iii) Stirrer (iv) Distilled water.

Procedure:

Step 1: Weigh 0.9 gm sodium chloride crystal by an analytical

balance.

Step 2: Take about 40ml distilled water into a 100ml volumetric

flask or a 100ml graduated measuring cylinder.

Step 3: Add the weighted sodium chloride crystal and allow to

dissolve using a stirrer.

Step 4: Add distilled water up to the mark 100ml.

100ml normal saline is prepared.

(b) Preparation of 100ml normal saline from 20% sodium

chloride Solution.

Principle:

V1S1 = V2S2

V₁ = Volume of normal saline to be prepared.

S₁ = Concentration of normal saline to be prepared.

15
 V2= Volume of supplied sodium chloride solution to be
used.

S2
Concentration of supplied sodium chloride solution to
be used.

VISI
V2

S2

Calculation:

ViSi
V2

S₂

Here, V₁ = 100ml; S₁ = 0.9%; V₂ = ?; S2 = 20%

100ml X 0.9%;

Therefore, V2 =

20%

=
: 4.5ml

So, Volume of 20% sodium chloride solution to be used =

4.5ml.

Apparatus & Requirements:

(i) Graduated glass pipette
(ii) 100ml volumetric flask or 100ml graduated
measuring cylinder
(iii) Stirrer
(iv) Distilled water.

Procedure:

Step 1: Pipette about 40ml distilled water into the 100ml

volumetric flask or 100ml graduated measuring cylinder.

Step 2: Add 4.5ml 20% sodium chloride Solution and allow to

mix.

16
Step 3: Add distilled water up to the mark 100ml.

100ml normal saline is prepared.

(c) Preparation of 100ml 5% dextrose solution from dextrose

powder.

Content: 100ml 5% dextrose solution contains 5.0 gm dextrose

powder.

Apparatus & Requirements:

(i) Analytical balance (ii) 100ml volumetric flask or
100ml graduated measuring cylinder (iii) Stirrer

Requirements: Distilled water.

Procedure:

Step 1: Weight 5.0 gm dextrose powder by an analytical balance.

Step 2: Take about 40ml distilled water into a 100ml volumetric

flask or a 100ml graduated measuring cylinder.

Step 3: Add the weighted dextrose powder and allow to dissolve

using a stirrer.

Step 4: Add distilled water up to the mark 100ml.

100ml 5% dextrose solution is prepared.

(d) Preparation of 100ml 5% dextrose solution from 25%

dextrose solution.

Principle:

V₁S1 = V₂S2

V₁ =
Volume of dextrose solution to be prepared.
S₁ = Concentration of dextrose solution to be prepared.
V2 = Volume of supplied dextrose solution to be used.
S2 = Concentration of supplied dextrose solution to be
used.

17
 VISI
V2

S2

Calculation:

ViSi
V2

S2
Here, V₁ 100ml; S₁ = 5.0%; V₂ = ?; S2₂ = 25%

100ml X 5.0%;
Therefore, V2 = =

25%

= 20ml

So, Volume of 25% dextrose solution to be used = 20ml.

Apparatus & Requirements:

(i) Graduated glass pipette
(ii) 100ml volumetric flask or 100ml graduated
measuring cylinder
(iii) Stirrer
(iv) Distilled water.

Procedure:

Step 1: Pipette about 40ml distilled water into the 100ml

volumetric flask or 100ml graduated measuring cylinder.

Step 2: Add 20ml 25% dextrose solution and allow to mix.

Step 3: Add distilled water up to the mark 100ml.

100ml 5% dextrose solution is prepared.

18
(d) Preparation of 100ml deci-normal (-------) hydrochloric acid
from 5.0 N hydrochloric acid.

Principle:

V1S1 = V2S2

V₁ = Volume of hydrochloric acid to be prepared.

S₁ Concentration of hydrochloric acid to be prepared.
=

V₂ = Volume of supplied hydrochloric acid to be used.

S₂ =
Concentration of supplied hydrochloric acid to be
used.

V₁S₁
V2

S2

Calculation:

V₁S₁
V2 =

S2
Here, V₁ 100ml; S₁ =
---; V₂ = ?;
‒‒‒‒‒--
S2 = 5.0 N

100ml X

Therefore, V2

5.0 N

= 2ml
=

So, Volume of 5.0 N hydrochloric acid to be used = 2.0 ml.

Therefore, distilled water required = (100-2)

= 98ml.

19
Apparatus & Requirements:
(i) Graduated glass pipette
(ii) 100ml volumetric flask or 100ml graduated
measuring cylinder
(iii) Stirrer

(iv) Distilled water.

Procedure:

Step 1: Pipette 98ml distilled water into the 100ml volumetric

flask or 100ml Graduated measuring cylinder.
Step 2: Add 2.0ml 5.0 N hydrochloric acid slowly by the side of
the apparatus.

100ml --=====

hydrochloric acid is prepared.

20
Identification of abnormal constituents of
urine.

A. Normal urine:

Urine is the excretedproduct of the body formed by the

kidneys. The daily urinary output is 600 to 2500ml
(average 1500ml). At nightly, volume of urine is usually
half of the daytime volume.

Composition:

Normal constituents of urine are

(i) Water- 94 - 96%
(ii) Solids - 4 - 6% (60gm/day)
(a) Organic substances: Urea, uric acid,
creatinine, hippuric acid, ascorbic acid, phenol etc.
(b) Inorganic substances: Na+, K+, Cl-, Ca++, PO4---
" SO4, Mg++, NH4+, etc.

Examination of urine: A) Physical B) Chemical C)

Microbiological
Physical Examination:

(i) Appearance: Clear and transparent.

(ii) Colour: Straw.
(iii) Odour: Ammonical
(iv) Specific gravity: 1.010- 1.030
(v)Osmolarity: 600-900 mosm/L
ChemicalExamination:

1) pH acidic with average pH of 6.4

2)Detection of abnormalconstituents ofurine (normally
absent with the exception of trace amount of protein)
Urine is said to be pathological when it contains abnormal
constituents.

21
 The abnormal constituents of urine with their cause of

appearance in urine & tests to detect them

Abnormal Condition Cause Biochemical

constituents test employed
Reducing Sugar Glycosuria: Diabetes mellitus, Benedicts test
(mainly glucose, Cushing's
sometime fructose, syndrome,
& galactose) Hyperthyroidism,
Acromegaly,
pheochromocytoma
Protein (mainly Proteinuria Nephrotic Heat

albumin) syndrome, Acute coagulation

test
glomerulonephritis,
toxemia of

pregnancy, severe
UTI, multiple
myeloma
Ketone bodies (B Ketonuria Prolonged Rotheras

hydroxyl butyric starvation, Nitroprusside

acid, acetoacetate, persistent test

etc.) vomiting, diabetic

ketoacidosis
Bile salt Obstructive Hays surface
(Na+ & jaundice tension test
K+taurocholate

& glycocholate)
Bile pigment Gmelins test

(Bilirubin,
Urobilinogen)
Blood Hematuria Renal stone, Benzidine test

tumour, trauma &

tuberculosis, AGN

22
 Tests for abnormal constituents of urine

Benedicts test: for detection of reducing substance

Experiment Observation Inference

5.0ml of Benedict's Blue colour of Reducing

solution is taken in Benedict's solution substance (sugar)
a test tube and changes to green, is present.
heated up to yellow, orange, red
boiling point. Then or brick red colour.
eight drop of Depending on the
supplied urine is concentration of
added and again reducing
heated up to substance.
boiling point.

2. Test for protein: Heat coagulation test:

Experiment observation Inference

(a) 2/3rd of a test tube is filled with (a) Turbidity

supplied urine, then the upper part appeared. Protein is

of urine is heated. present.

(b)Few drops of glacial acetic acid is (b) Turbidity (albumin)
added and heated again. intensified.

23
 3. Test for ketone bodies: Rothera'snitroprusside test:

observation Inference
Experiment
Ketone
5ml of urine is taken in a test A

tube and saturated with crystals permanganate body is

of ammonium sulfate. Then 3 coloured ring present.
drops of freshly prepared 5% appeared at
sodium nitroprusside solution is the junction
added and shaken well. Then of two layer.
2.0ml of concentrated ammonium

hydroxide is added slowly by the

side of the test tube.

4. Test for bile salt: Hay's surface tension test:

observation Inference
Experiment
2.0ml of urine and 2.0ml of distilled Sulfur powder Bile salt is
water is taken in two separate test sinks in urine present.
tube. Then trace amount of sulfur but not in

powder is added. water.

24
 5. Test for bile pigment: Gmelin's test:

Experiment observation Inference

2.0ml of concentrated nitric A red/blue/green/violet/ Bile

acid is taken in a test tube. reddish blue coloured pigment is
Then 5.0ml of urine is added ring appeared at the present.
slowly by the side of the test junction of two layer.
tube.

6. Test for blood: Benzidine test:

Experiment observation Inference

3.0ml of glacial Violet colour remains. Blood is absent.

acetic acid is taken

in a test tube and Violet colour changed to Slight amount of
saturated with blue. Blood is present.
Benzidine powder;
then 2.0ml of urine Violet colour changed to Moderate amount
and 1.0ml of 3% green. of Blood is

hydrogen peroxide present.

is added and mixed.

Violet colour changed to Severe amount of

black. Blood is present.

25
 Study of Photometry

Light: Light is the visible spectrum of electromagnetic radiation, emitted in the form
of waves of different wave lengths ranging from 380nm to 750nm.

Visible

Light
700nm 600nm 500nm 400nm

Radio waves Microwaves

Thi
Infrared Ultraviolet X-rays Gamma

wwwwwwww
LONGER
WAVELENGTH (meters) SHORTER
T T T
10¹

10² 1¹
1 10-² 10 10- 10-9 10-5 10-7 10-ª 10-9 10-30 10-11 10-12 10-15

Dispersion of light

White light (stray light) is the composite mixture of all the wave length of
visible spectrum of electromagnetic radiation

When light is passed through a dispersion medium like prism, then we will get
a light of a particular wave length.

Types of light

A. Stray light/ Polychromatic light

B. Monochromatic light

Common dispersion media

■ Filter

Prism

▪ Diffraction grating
▪ Interference filter

How we see different color

■ If light falls on a substance and totally absorbed, it will be visualized as black.

If the light is totally reflected, it will visualized as white.

26
 ▪ If some portion of light are absorbed and some are reflected, the reflected wave

length will determine the color of substance

Wave length: The distance between two adjacent peaks or two adjacent crests of two
adjacent waves is called wave length.

Wave
Wavelength

Distance

Frequency: It is the number of waves (or, wave motion) (transmitted) per second.

The more the wave lengths, the less will be the frequency. The more the frequency,

the more will be the penetrating power.

Long Short
Wave Wave

Length Length

wwww
Low Frequency High Frequency

Low Energy High Energy

Photometry: Photometry is a technique used to measure the concentration of a

substance in a solution by adopting the property of absorption or transmission or

emission of light of a particular wave length by that substance present in the solution

under controlled condition. Or, Photometry is the procedure to measure the intensity

of light.

27
Principles of Photometry:

1. Substance to be measured by photometry must be colored to begin with (like

Hemoglobin) or can be made to produce color derivatives by using certain
reagents and reactions (e.g. Glucose, Urea etc.)
2. Intensity of color produced is proportional to the concentration of the color

producing substance present in the solution.

3. Colored substance absorbs light of a particular wave length and the extent of
light absorption depends on the concentration of color producing substance in
the solution.

Instruments follow the principles of photometry:

1. Colorimeter

2. Spectrophotometer
3. Fluorometer

4. Flame photometer

5. Atomic absorption spectrophotometer

6. Electro photometric and chromatographic scanner.

Laws of Photometry:
Beer's law: This law states that, the intensity of transmitted light decreases

exponentially with the increase in concentration of substance in the solution.

Or, absorbance is proportional to concentration of coloured substance in a solution.

Lambert's law: This law states that, the intensity of transmitted light decreases

exponentially with the increase in the length of light pathway. (This law is nullified as

the cuvet is unchanged)

Or, absorbance is proportional to path length of the solution.

Io I

Incident Light Transmitted Light

28
 Now, using photometric laws, we can write
A∞ C x L
Here, A= absorbance

A = K xC XL
K= proportionate constant
K=A/CXL
C= concentration of substance

L= length of light pathway

For standard solution (s), For unknown solution (u),
we can write we can write

Ks = Aş/ Cs x Ls Ku=Au /Cux Lu

"K" is a photometric constant and is fixed for a particular substance whatever may be
the concentration. "L" is also kept constant by using the cuvette having same diameter
or length of light pathway.

So, we can write, Ks = Ku

So, As/ Cs x Ls = Au / Cu x Lu

Cu = Aux Cs / As [as Ls=Lu, because of using cuvette of same length of light

pathway]
Absorbance of Unknown X Concentration of Standard
Concentration of Unknown =

Absorbance of standard

Colorimeter

It is an instrument by which color is measured or compared. Thus colorimeter

employs color and color variation to determine the concentration of substances in

solution

Figure: Colorimeter

29
Parts of a Colorimeter

The major components of a photometer are shown schematically in figure. These

include

(a) Light source (b) Entrance slit (c) Monochromator or filter (d) Exit slit (e) Cuvette
(f) Photodetector (g) Meter.

Light Entrance Monochromator Exit Cuvet Detector Meter

source slit
slit

Figure: Parts or components of a colorimeter

(a) Light source: Usually tungsten lamps are used. Tungsten lamp is acceptable for
measurement of moderately dilute solutions in which the change of absorbance varies
significantly with small change in concentration.

(b) Entrance slit: The entrance slit is a narrow slit by which way the light beam passes
to the monochromator.

(c) Monochromator: The monochromator passes the light beam to one selected
wavelength ray. The simplest type of monochromator is a thin layer of coloured glass,
also known as filter.

(d) Exit slit: The exit slit is another narrow slit by which the ray of one selected
wavelength passes to the cuvette.

(e) Cuvette: Cuvette is a small vessel used to hold a liquid sample to be analyzed in

the light path of a spectrophotometer. Cuvette is also known as the absorption cell.

Cuvette may be round, square or rectangular and constructed from glass, quartz or

polystyrene. Cuvette contains solution the absorbance of which is to be measured.

(f) Photodetector: Photodetector is a device that converts the ray energy into an

electrical signal.

30
(g) Meter: Meter indicates the absorbance or transmittance of the solution in the
cuvette. The meter can be displayed digitally or by analogue indicator.

Differences between Colorimeter and Spectrophotometer:

Traits Colorimeter Spectrophotometer

Monochromator Filter Prism

Spectral bandwidth Broad band Narrow band

Spectral purity Less More

Spectral isolation Filter has to be changed Desired wave length can be

with different analysis adjusted

Stray light High degree/ more Minimum

Accuracy Less More

Sensitivity Less More

Specificity Less More

Calibration Manual Auto-calibrated

Sample volume Larger volume needed Small volume needed

Cost More costly

Cheaper

Range of electromagnetic Only visible range of light Beyond the visible range of
radiation can be used light can be used

31
 Estimation of Blood Glucose

Method: Glucose Oxidase method.

Principle: Glucose is oxidized by glucose oxidase to produce gluconic acid and

hydrogen peroxide.

Glucose Oxidase
Glucose + 02 Gluconic acid +H₂O2

The formed hydrogen peroxide then reacts with Phenol and 4-Amino phenazone to
produce a coloured complex, Quinonemine by another enzyme peroxidase.

Peroxidase
H₂O2 + Phenol + 4-Amino phenazone →Quinonemine + H₂O

The intensity of colour produced is proportional to the glucose concentration in the

sample.

Instruments: (a) Centrifuge machine

(b) Photoelectric colorimeter
(c) Three test tubes
(d) Micropipettes

Reagents: Complete test kit for glucose.

Contents

(a) Phosphate buffer at pH 7.4

(b) Glucose Oxidase
(c) Peroxidase
(d) Phenol
(e) 4-Amino phenazone

Standard Glucose solution: Concentration is 5.5mmol/l.

Specimen: Blood.

Procedure:

Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then separate
the serum.

Step2: Take three test tubes and mark as 'Blank', 'Standard' and 'test'. Pipette
1.0 ml of reagent to each of the three test tubes.
Step3: Add 10μl of standard glucose solution to the 'Standard' test tube and 10μl of
serum to the 'test' test tube and mix.

Step 4: Incubate all the three test tubes at room temperature for ten minutes.
Step 5: Read the absorbance of the contents of 'Standard' test tube and the
contents of 'test' test tube against blank by a colorimeter at 530nm wavelength.

32
Calculation:

Absorbance of test:
Absorbance of standard:
Concentration of standard: 5.5mmol/l.
Absorbance of test
Blood Glucose Concentration = X Concentration of
standard.

Absorbance of standard

Result: Glucose level of the supplied blood is mmol/l.

Normal range: Fasting- 3.9 to 6.0 mmol/l.

Random- 4.7 to 8.8 mmol/l.

Note: Conversion factor for glucose:

From SI unit to traditional unit is

mmol/IX 18

From traditional unit to SI unit is

mg/dl / 18

33
 Estimation of SerumTotal Cholesterol

Method: Enzymatic method.

Principle: Cholesterol and its estersare released from lipoproteins by detergents.

Cholesterol ester is hydrolyzed by cholesterolesteraseto produce cholesterol and
free fatty acid.

Cholesterolesterase

Cholesterol ester + H₂O →Cholesterol + FFA

Cholesterol is oxidized by cholesterol oxidase to produce Cholestene-3, land

hydrogen peroxide.
Cholesterol Oxidase

Cholesterol + O2 →Cholestene-3,1+ H₂O2

The formed hydrogen peroxide then reacts with Phenol and 4-Amino phenazone to
produce a coloured complex, Quinonemine by another enzyme peroxidase.

Peroxidase

H₂O2+ Phenol + 4-Amino phenazone Quinonemine + H₂O

The intensity of colour produced is proportional to the totalcholesterol

concentration in the sample.

Instruments:

(a) Centrifuge machine

(b) Photoelectric colorimeter
(c) Three test tubes
(d) Micropipettes

Reagents:

Complete test kit for cholesterol.

Contents

(a) Phosphate buffer at pH 6.9

(b) Cholesterolesterase
(c) Cholesterol Oxidase
(d) Peroxidase
(e) Phenol
(f) 4-Amino phenazone

Standard cholesterol solution: Concentration is 200mg/dl.

Specimen: Blood.

Procedure:

34
Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then
separate the serum.
Step 2: Take three test tubes and mark as 'Blank', 'Standard' and 'test'. Pipette
1.0 ml of reagent to each of the three test tubes.
Step 3: Add 10μl of standard cholesterol solution to the 'Standard' test tube
and 10 µl of serum to the 'test' test tube and mix.
Step 4: Incubate all the three test tubes at room temperature for ten minutes.
Step 5: Read the absorbance of the contents of 'Standard' test tube and the
contents of 'test' test tube against blank by a colorimeter at 530nm wavelength.

Calculation:

Absorbance of test:
Absorbance of standard:

Concentration of standard: 200mg/dl.

Absorbance of test
TotalCholesterol Concentration X Concentration of
standard.

Absorbance of standard

Result: Serum totalcholesterol level of the supplied blood is mg/dl.

Normal range: 150 to 220mg/dl.

Note: Conversion factor for cholesterol:

From SI unit to traditional unit is

mmole/liter X 38.7

From traditional unit to SI unit is

mg/dl/ 38.7

35
Estimation of Serum Total Protein

Method: Biurate method

Principle: Determination of the total protein is based on the principle of Biurate

reaction. Biurate reagent is copper salt in an alkaline media. Protein forms a blue
colored complex when treated with cupric ions in alkaline solution. The color
concentration of formed blue color is proportional to the protein concentration.
Instruments: (a) Centrifuge machine
(b) Photoelectric colorimeter
(c) Three test tubes
(d) Micropipettes

Reagents: Biurate reagent

Contents
(a) Sodium iodide.
(b) Potassium-sodium tartarate
(c) Copper sulfate
(d) Potassium iodide
dl.

Specimen: Blood.

Procedure:

Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then
separate the serum.
Step 2: Take three test tubes and mark as 'Blank', 'Standard' and 'test'. Pipette
1.0 ml of reagent to each of the three test tubes.
Step 3: Add 25μl of standard Proteinsolution to the 'Standard' test tube and 25μl
of serum to the 'test' test tube and mix.

Step 4: Incubate all the three test tubes at room temperature for ten minutes.
Step 5: Read the absorbance of the contents of 'Standard' test tube and the

contents of 'test' test tube against blank by a colorimeter at 530nm wavelength.

Calculation:
Absorbance of test:

Absorbance of standard:

Concentration of standard: 5.0gm/dl.

Absorbance of test
Serum Total Protein Concentration X Concentration of
standard.

Absorbance of standard

Result: Total Proteinconcentration of the supplied blood is gm/dl

Normal range: 6.0-8.0 gm/dl

36
 Estimation of Blood Urea

Method: Urease/Enzymatic method.

Principle:
Urea + H₂O
Urea is hydrolyzed by urease to produce ammonia and carbon-di-oxide.
2NH3 + CO2
The formed ammonium ions then reacts with salicylic acid and hypochlorite to give
a green colored dye, 2,2 dicarboxy indophenol. The intensity of color produced is
proportional to ureaconcentration in the sample.
Instruments: (a) Centrifuge machine
(b) Photoelectric colorimeter
(c) Three test tubes
(d) Micropipettes

Reagents: Reagent 1: Buffer reagent

Contents

(a) Phosphate buffer at pH 6.7

(b) EDTA
(c) Sodium salicylate
(d) Sodium nitroprusiate
Reagent 2: Enzyme reagent
Contents

(a) Urease

Reagent 3: Hypochlorite solution

Contents

(a) Sodium hypochloride

(b) Sodium hydroxide
Standard ureasolution: Concentration is 50mg/dl.

Reagent Preparation: Mix reagent 1 and reagent 2 at a ratio of 20 : 1 prior to use.

This solution will known as working reagent.
Specimen: Blood.

Procedure:

Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then
separate the serum.
Step 2: Take three test tubes and mark as 'Blank', 'Standard' and 'test'. Pipette
1.0 ml of workingreagent to each of the three test tubes.
Step 3: Add 10μl of standard urea solution to the 'Standard' test tube and 10 μl
of serum to the test' test tube and mix.
'

Step 4: Incubate all the three test tubes at room temperature for five minutes.
Step 5: Then add 1.0 ml of reagent 3 and mix.
STEP 6: Incubate all the three test tubes at room temperature for ten minutes.
Step 5: Read the absorbance of the contents of 'Standard' test tube and the
contents of 'test' test tube against the contents of 'blank'test tube by a colorimeter
at 530nm wavelength.

37
Calculation:
Absorbance of test:
Absorbance of standard:
Concentration of standard: 50 mg/dl.
Absorbance of test
Blood Urea Concentration =
X Concentration of standard.
Absorbance of standard

Result: Blood Urea Concentration of the supplied blood is mg/dl

Normal range: 15-50 mg/dl

Note: Conversion factor for Urea:

From SI unit to traditional unit is
mmol/1X 6

From traditional unit to SI unit is

mg/dl / 6

38
 Estimation of Blood Total Bilirubin
Method: DMSO(dimethyl sulphoxide) method.
Principle:Bilirubins and diazonium salt of sulfanilic acid produces azobilirubin in
acid medium, which shows maximum absorption at 555nm. The intensity of the
color produced is proportional to the quantity of bilirubins which has reacted. In
the absence of an accelerator only conjugated bilirubins react. In the presence of
an accelerator, the dimethyl sulphoxide (DMSO) the nbon-conjugated bilirubin also
participates to the reaction, thus to determine the level of total bilirubin.
Instruments: (a) Centrifuge machine
(b) Photoelectric colorimeter
(c) Two test tubes
(d) Micropipettes

Reagents: Reagent 1: DMSO reagent

Contents

(e) Sulfanilic acid

(f) HC1
(g) Sodium Chloride
(h) Ethylene glycol
(i) DMSO

Reagent 2: Sample blank reagent

(a) Sodium Chloride
Reagent 3: Nitrite reagent
Contents

(a) Sodium nitrite.

Specimen: Blood.

Procedure:

Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then
separate the serum.

Step 2: Take two test tubes and mark as 'Sample Blank' and TestSample'.
Step 3: Pipette 1.0 ml of reagent 1 to the Test Sample' test tube and 1.0 ml of
reagent 2 to the 'Sample Blank' test tube.

Step 4: Add 100μl of Reagent 3 to both the test tube.

Step 5: Add 100μl of serum to both the test tubeand mix.

Step 6: Incubate both the test tubes at room temperature for five minutes.
Step 7: Read the absorbance of the contents of Test Sample'test tube against the
contents of 'Sample Blank'test tube by a colorimeter at 530nm wavelength.

39
Calculation:

Absorbance of test against blank:

dl.

Result: Total Bilirubin Concentration in blood =

mg/dl.

Normal range: 0.2 -- 1.2mg/dl.

Note: Conversion factor for Bilirubin:

From SI unit to traditional unit is
micromole/liter / 17.1

From traditional unit to SI unit is

mg/dlX 17.1

40
 Estimation of Serum Creatinine

Method: Fixed time kinetic method.

Principle: Colorimetric reaction of creatinine with picrate is based on Jaffe

reaction. Creatinine reacts with alkaline picrate forming a red coloured complex.
This reaction is measured kinetically without any pretreatment step.

Instruments: (a) Centrifuge machine

(b) Photoelectric colorimeter
(c) Stop watch
(d) Micropipettes

Reagents: Reagent 1 (picricacid reagent)

Contents

(a) Sodium dodecyl sulfate.

(b) Picric acid

Reagent 2 (Base reagent)

Contents:

(a) Sodium hydroxide

(b) Disodium phosphate
(c)
Standard creatinine solution: Concentration is 2.0mg/dl

Reagent Preparation: Mix reagent 1 and reagent 2 at a ratio of 1:1 prior to use.
This solution will known as working reagent.

Specimen: Blood.

Procedure:

Step 1: Centrifuge the supplied blood at 2500 r.p.m. for ten minutes, then separate
the serum.

Step 2: Pipette 1ml of working reagent into a cuvette. Then add 100μl standard
creatinine solution and mix. Immediately start the stopwatch and read the
absorbance at 30sec (As30) and 120sec (As120) by a colorimeter at 530nm
wavelength.

Step 3: Pipette 1ml of working reagent into another cuvette. Then add 100μl
serumand mix. Immediately start the stopwatch and read the absorbance at 30sec
(AT30) and 120sec (AT120) by a colorimeter at 530nm wavelength.

41
 Calculation:

Initialabsorbance of test at 30sec (AȚ30)

Final absorbance of test at 120sec (AT120)
Change of absorbance of test: (AT120) - (AT30)
Initialabsorbance of standard at 30sec (As30)
Final absorbance of standard at 120sec (As120)
Change of absorbance of Standard: (As120) - (As30)
Concentration of standard: 2.0mg/dl

Change of absorbance of test

Serum creatinine Concentration= XConcentration of standard
Change of absorbance of Standard

Result: CreatinineConcentration of the supplied blood is mg/dl

Normal range: 0.6-1.2 mg/dl

Note: Conversion factor for Creatinine:

From SI unit to traditional unit is
micromole/liter/ 88.4

From traditional unit to SI unit is

mg/dl X 88.4

42