DATE:

Exp No: 1(a) GENERAL TESTS FOR CARBOHYDRATES

AIM:

To identify and analyze the given test sample.

S. EXPERIMENT OBSERVATION INFERENCE

NO

1 SOLUBILITY TEST:

Note the Solubility of the given Completely soluble in

test sample. Water.

Insoluble in Water.
2 MOLISCHS TEST:

Add 2 drops of Molischs A red violet ring is

reagent (15% 1-Naphthanol in
alcohol) to about 2 ml of test
solution and mix well. 2 ml of
Conc. Sulphuric acid is added
along the sides of the test tube.
formed at the junction of
two layers.
No characteristic change.

3 IODINE TEST:

To 2 ml of sugar solution add Appearance of deep blue

few drops of 0.01N Iodine colour.
solution in KI.
Appearance of brown
colour.

No characteristic colour
change.

4 BENEDICTS TEST:

To 2ml of sugar solution add Formation of brick red,

equal volume of Benedicts yellow or green colour
reagent and boil for 5 minutes precipitate.
in a water bath. Cool the
solution. No characteristic colour
(or) precipitate is
obtained.

1
FLOWCHART

2
5 FEHLINGS TEST:

Add equal volume of Fehlings Formation of yellow (or)

A and B . Take 5 ml of this orange red colour
solution and add 2 ml of sugar precipitate
sample , and heat in a water
bath for 5 minutes and allow it No characteristic colour
to cool. or precipitate is obtained

6 BARFOEDS TEST:

To 5 ml of Barfoeds solution Formation of brick red

add 0.5 ml sugar solution and precipitate
heat in water bath for a few
minutes. No characteristic colour
precipitate is obtained

7 SELIWANOFFS TEST:

To 2 ml of seliwanoff reagent Appearance of deep red

add 2 drops of test solution and colour (cherry red colour)
heat the mixture for 10 minutes.

No characteristic colour
is obtained

8 BIALS TEST:

To 2 ml of the given solution Appearance of green

add equal volume of Bials colour precipitate
reagent and heat in water bath
and cool it.
No characteristic colour
or precipitate is obtained

3
4
 9 OSAZONES TEST:

To 2 ml of sugar solution add Yellow needle shaped

0.5 g of phenyl hydrazine crystal within 3-5
hydrochloride, 1 gm of sodium minutes.
acetate & ten drops of glacial
acetic acid is added and heated Yellow needle shape
in a boil water bath for half-an- crystal within 8-12
hour. Allow it to cool slowly to minutes.
examine the crystal under Absence of crystal
microscope. formation.

Note:

OSAZONE SOLUBILITY IN OSAZONE

CARBOHYDRATE BOILING WATER CRYSTAL
COMPOUND
Glucose Glucosazone Insoluble Bundle of grass

Mannose Mannosazone Insoluble Bundle of grass

(bloom-like)

Fructose Fructosazone Insoluble Bundle of grass

(hay-like)

Galactose Galactosazone Insoluble Needle shaped

Maltose Maltosazone Soluble Sunflower shaped

Lactose Lactosazone Soluble Puff ball shaped

RESULT:

5
6
 DATE:

Exp No: 1(b) GENERAL TESTS FOR LIPIDS

AIM:

To identify and analyze the given test solution.

S.NO EXPERIMENT OBSERVATION INFERENCE

1 SOLUBILITY TEST:

Add 5 drops of test Oil in a) In water tube, the water

test tubes containing water,
dilute acetic acid, dilute
sodium hydroxide, alcohol,
Chloroform, Benzene and
Carbon Tetra Chloride .
Shake vigorously and allow
standing. Note solubility in
each tube.
separates quickly
b) Insoluble in inorganic
solvents.
c) In Chloroform tube,
soluble in chloroform.

2 GREASE SPOT TEST:

Take 3 ml of Ether in a test Ether evaporates leaving a

Tube. Dissolve 5 drops of oil translucent spot on the filter
in it. Put a drop of this paper.
solution on a filter paper and
let it dry. No characteristic change.

4 ACROLIEN TEST:

In an absolutely dry test tube, Typical pungent odour is

pour two drops of test sample. produced.
Add a pinch of Potassium
bisulphate (KHSO4) , Heat No characteristic change.
gently the test tube over a
small flame.

7
8
5 SAPONIFICATION TEST:

Take 4ml of 2% sodium

carbonate solution in a test
tube and add two drops of
Mustard oil. Shake vigorously
and boil. A clean soapy
solution is formed cool and
divide it into three parts for
the following test:
The separated Fatty acids
In one test tube, add a few
floating are observed.
drops of conc. HCl.
White precipitate of soap
In second test tube, dissolve
seperate out and float on the
sufficient amount of finely
surface.
powdered NaCl.
A precipitate of insoluble
In the third test tube, add
calcium soap is obtained.
small amount of CaCl2.

6 TEST FOR DOUBLE

BOND :
At first, orange color
Dissolve the given oil sample develops and then
in ethanol and add 0.4 % decolourizes.
bromine in ethanol, drop by
drop .
7 SALKOWSKI TEST:

Dissolve a few crystals of The color in upper

.Cholesterol in chloroform and
then add equal amount of
conc. Sulphuric acid by the
side of the test tube.
chloroform layer changes
from bluish - red to cherry
red and purple whereas
lower acid layer develops
green color.

9
10
8 LIEBERMAN-
BURCHARD TEST:

Take 3 ml of given test

solution, add 10 drops of A red color is formed which
acetic anhydride and 1 to 3 turns blue and finally bluish
drops of conc.H2SO4. green.

9 ZACK'S TEST:

To 2 ml of the test solution , Red colour develops.

add 3 ml of ferric chloride in
acetic acid and add 2 ml of
conc. H2SO4.

RESULT:

11
12
DATE:

Exp No: 1 (c) GENERAL TEST FOR PROTEINS

AIM:

To analyze and identify the presence of protein in the given sample .

S.NO EXPERIMENT OBSERVATION INFERENCE

1 NINHYDRIN TEST:

To 1ml of given solution add 2 Blue/Purple color

drops of freshly prepared 0.1% develops.
solution of Ninhydrin. Heat it in a
boiling water bath. No blue color
develops

2 BIURETS TEST:

To 2 m1 of test sample add 2 ml Pink, Purple or blue

of 10% NAOH mix well and add colour or precipitate
0.5 % CuSO4 solution drop by develops
drop.
No color develops

3 XANTHOPROTEIC TEST:

To 5 ml of protein solution A white precipitate

carefully add 1 ml of Conc. HNO3 which changes yellow
. Boil it for 1 minute , cool the on heating and
test tube under tap water and add develops to orange on
excess of 40% NaOH or ammonia addition of NaOH.
.
No white precipitate is
formed

4 SAKAGUCHI TEST:

To about 3 ml of protein solution Intense red color

add 1 ml of Sakaguchi reagent( develops.
5% NaOH and 2 drops of
alcoholic -Napthanol) and cool. No red color develops.
Add few drops of bromine water.

13
14
5 SULPHUR TEST:

To 2 ml of test solution , add A brown or black precipitate

2 ml of 40% NaOH and 10 is formed.
drops of lead acetate
solution. Boil for a minute
and cool it. No brown or black
Precipitate is formed.

6 MOLISCHS TEST:

To 3 ml of test solution add A reddish violet ring is

1 ml of Molischs reagent
and then concentrated
sulphuric acid is added
along sides of test tube.
formed at the junction of two
lipids.
No violet ring is formed.

7 TEST FOR ORGANIC

PHOSPHATE:

To 2 ml of given solution,
Yellow colour develops
add 1 ml of 40% NaOH.
Heat strongly for 2-3 No characteristic change.
minutes and then cool. Add
0.5ml of conc. HNO3 and
filter. To the filtrate, add a
pinch of ammonium
8 molybdate
MILLON'Sand warm gently
TEST:

To 5ml of protein solution A white precipitate is formed

add a few drops of freshly which turns red upon
prepared Millon's reagent heating.
and heat to boiling.

RESULT:

15
16
DATE:

Exp No: 2 PREPARATION OF SERUM AND PLASMA FROM BLOOD

AIM:

To prepare serum and plasma from blood.

THEORY:

Blood plasma is the liquid component of blood, in which the blood cells are suspended. It
makes up about 60% of total blood volume. It is composed of mostly water (90% by volume), and
contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide
(plasma being the main medium for excretory product transportation). Plasma is the supernatant
fluid obtained when anti-coagulated blood has been centrifuged. The blood is mixed with an
appropriate amount of anticoagulant like heparin, oxalate or ethylene diamine tetraacetic acid
(EDTA). This preparation should be mixed immediately and thoroughly to avoid clotting. Blood
serum is blood plasma without fibrinogen or the other clotting factors. Serum is clearer than
plasma because of fewer proteins. Proteins are sometimes considered as interfering substances in
some tests as they react with the reagent and thereby yield inaccurate results. Serum is the
preferred specimen in clinical testing as the interference that may be caused by a plasma specimen
because of the presence of an anticoagulant, is eliminated. If storage is necessary prior to
processing, store the blood at room temperature, shielded from light, and on a rocker.

BLOOD PLASMA PREPARATION

Materials and Equipments

Human blood sample.

Vacutainer tubes containing anticoagulant (BD Vacutainer plastic EDTA tube, 10 ml)
Serological pipettes of appropriate volumes (sterile)
Centrifuge tubes
Cryovials
Benchtop centrifuge (NOT refrigerated) with swing-out rotor and appropriate carriers

PROCEDURE

1. Draw blood into vacutainer tube(s) containing ~1.8 mg K2EDTA per ml blood (may vary
depending on manufacturer). Be sure to draw the full volume to ensure the correct blood-
anticoagulant ratio.

2. Invert vacutainer tubes carefully 10 times to mix blood and anticoagulant and store at room
temperature until centrifugation.

3. Samples should undergo centrifugation immediately. This should be carried out for a minimum
of 10 minutes at 1000-2000 RPM (generally 1300 RPM) at room temperature

17
18
4. This will give three layers: (from top to bottom) plasma, leucocytes (buffy coat), and
erythrocytes.

5. Carefully aspirate the supernatant (plasma) at room temperature and pool in a centrifuge tube.
Take care not to disrupt the cell layer or transfer any cells.

6. Inspect plasma for turbidity. Turbid samples should be centrifuged and aspirated again to
remove remaining insoluble matter.

7. Aliquot plasma into cryovials and store at 80 C. Ensure that the cryovials are adequately
labeled with the relevant information, including details of additives present in the blood.

BLOOD SERUM PREPARATION

Materials and Equipment

Human blood sample.

Vacutainer tubes (containing either no additive or a clot activator)
Clot activator and silica gel.
Serological pipettes of appropriate volumes (sterile)
Centrifuge tubes
Cryovials
Benchtop centrifuge (NOT refrigerated) with swing-out rotor and appropriate carriers

PROCEDURE

1. Draw whole blood into vacutainer tube(s) containing no anticoagulant. Draw approximately 2
times the volume needed for use e.g. 10 ml blood for 4 ml serum.

2. Incubate in an upright position at room temperature for 30-45 min (no longer than 60 min) to
allow clotting. If using a clot-activator tube, invert carefully 5-6 times to mix clot activator and
blood before incubation.

3. Centrifuge for 15 min at manufacturers recommended speed (usually 1000-2000 RPM). Do not
use brake to stop centrifuge. Carefully aspirate the supernatant (serum) at room temperature and
pool into a centrifuge tube, taking care not to disturb the cell layer or transfer any cells. Use a
clean pipette for each tube.

4. Inspect serum for turbidity. Turbid samples should be centrifuged and aspirated again to remove
remaining insoluble matter. Aliquot into cryovials and store at 80 C. Ensure that the cryovials
are adequately labeled with the relevant information, including details of additives

RESULT:

19
 GLUCOSE ESTIMATION

MODEL GRAPH:
A standard graph is drawn with optical density in Y axis vs concentration of glucose in X axis.
The amount of glucose present in the given blood sample is then calculated.

Y axis

A T

OD at 510 nm

Concentration of Glucose (g/dl)

20
DATE:

Exp No: 3 ESTIMATION OF BLOOD GLUCOSE BY GLUCOSE OXIDASE- PEROXIDASE

METHOD

AIM:

To estimate the amount of glucose present in the given blood sample by Glucose Oxidase-
Peroxidase method.

PRINCIPLE:

Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide .
The formed hydrogen peroxide (H2O2) further reacts with phenol and 4- aminoantipyrine by the catalytic
action of peroxidase (POD) to form a red colored quinonimine dye complex . The intensity of the color
formed is directly propotional to the amount of glucose in the sample.

-D-glucose Mutarotase -D-glucose

-D-glucose +H2O+O2 Glucose oxidase D-gluconic acid+ H2O2

H2O2+ 4-Aminophenazone+phenol Peroxidase Quinonemine + 4 H2O

CLINICAL SIGNIFICANCE:

The requirement for serum glucose level analysis is made in case of suspected diabetes mellitus
(DM) .Increased serum glucose level is hyperglycemia with glycosurea is a diagnosis for diabetes mellitus.
In this case of glucose tolerance test (GTT) and increased level of glucose is also noted with increased
endocrine activity. A decreased level of serum glucose in hypoglycemia is often associated with starvation
and hyperinsulinism, and decreased endocrine activity.

Several methods have been for glucose determination such as:

Benedict's quantitative method

O-toluidine method
Glucose oxidase method
Alkaline copper reduction method

REAGENTS REQUIRED:

Solution 1: Glucose Enzyme Reagent

Solution 2: D-Glucose Stock standard solution (1 mg/ml)
Solution 3: D-Glucose Working standard solution

21
OBSERVATION TABLE:

Addition Sequence Blank Standard Test

Enzyme Reagent (ml)
Standard (ml) - -
Sample (ml) - -
Optical Density/
Absorbance at 505 nm

CALCULATION:

Concentration of Glucose (mg/dl) =

22
 PROCEDURE

Preparation of Blood Sample

0.2ml of blood sample is taken in a centrifuge tube. To this 0.3ml of 10% sodium tungstate,
0.3ml of 2 / 3N sulphuric acid and 3.2ml of distilled water are added to precipitate the proteins. It is kept
aside for 10 minutes and then centrifuged at 3000 rpm for 10 min. 1 ml of the supernatant is taken for the
estimation of glucose.

Estimation of glucose

1 ml of Enzyme reagent is pipetted out into clean dry test tubes labelled as blank (B), Standard (S) and
Test (T) .10 l of Standard and Test sample is taken in respective test tubes (i.e S & T) . All the test tubes
are heated for 20 minutes in a boiling water bath at 40 50oC. Absorbances are read at 505 nm against the
reagent blank .

RESULT:

23
CREATININE ESTIMATION

OBSERVATION TABLE:

PARTICULARS BLANK STANDARD TEST

Creatinine Standard (ml)

Test Sample (ml)

Distilled Water (ml)

0.75 N NaOH (ml)

Saturated Picric Acid (ml)

Mix well and incubate at room temperature for 15 minutes

Optical Density at 540 nm

24
DATE:

Exp No. 4 ESTIMATION OF CREATININE IN URINE

AIM:

To estimate the amount of Creatinine present in the given urine sample by Jaffe's Alkaline Picrate
Method.

PRINCIPLE:

Creatinine develops an orange red colour when treated with picric acid in the presence of
strong alkali. This colour is due to the formation of creatinine picrate and measured at 540 nm.
Normal adults excrete creatinine from 1-1.8 mg/day. Under heavy meat diet, wasting disease,
prolonged starvation, fever etc., it increases.

CLINICAL SIGNIFICANCE:

A Creatinine test reveals important information about the kidneys. Creatinine is a chemical waste
product that's produced by muscle metabolism and to a smaller extent by eating meat. Healthy kidneys
filter creatinine and other waste products from blood. The filtered waste products leave the body in
urine. If kidneys aren't functioning properly, an increased level of creatinine may accumulate in the
blood. A serum creatinine test measures the level of creatinine in the blood and gives an estimate of how
well the kidneys filter (glomerular filtration rate). A creatinine urine test can measure creatinine in
urine.

REAGENTS REQUIRED:

1. Creatinine standard solution

Dissolve 100 mg creatinine in 100 ml of 0.1 N HCl. Working standard solution may be prepared
by appropriate dilution of the stock solution.
2. Saturated picric acid solution (about 1%)
3. Sodium hydroxide solution (0.75 N )
4. Hydrochloric acid solution ( 0.1N)
5. Sodium tungstate (10%)
6. Sulfuric acid (2/3 N)

Preparation of protein free filtrate: To 1 ml urine/blood sample, add 8 ml distilled water, 0.5 ml
of 2/3 N sulfuric acid and 0.5 ml of 10% sodium tungstate solution in a stoppered centrifuge tube and
mix the contents. Then centrifuge at 3000 rpm for 10 min and collect the supernatant.

25
CALCULATION:

26
PROCEDURE:

1. Dilute 1 ml of urine in the ratio of 1:100

2. Pipette out 2.5 ml of working standard in to the labeled test tube as S.
3. Pipette out 4 ml of the given urine sample in another test tube labelled T.
4. Make up the volume to 4 ml in all the test tubes. A tube with 4 ml of distilled water serves as the
blank.
5. Now add 1 ml of saturated picric acid solution and 1 ml of 0.75 N NaOH. Mix the contents of the
tubes and incubate at room temperature for 15 min.
6. Then read the absorbance at 540 nm against blank.
7. Then plot the standard curve by taking concentration of creatinine along X-axis and absorbance at
540 nm along Y-axis.
8. From this standard curve calculate the concentration of creatinine in the given sample.

NORMAL RANGE:

Creatinine 15 25 (mg creatinine per 24 hours / Kg body wt)

RESULT:

27
UREA ESTIMATION

OBSERVATION TABLE:

Reagent Test Standard Blank

Working Reagent 1 ml 1 ml 1 ml
Standard - 10l -
Sample - - 10 l
Distilled water 2ml 2ml 2ml
Mix well and Incubate at 37oC for 5 minutes
Chromogen Reagent 1 ml 1 ml 1 ml
o
Mix well and Incubate at 37 C for 5 minutes
Optical Density at 600 nm

CALCULATION:

REFERENCE VALUE:
(To convert from mmol/l to mg/dl, multiply by 6. To convert from mg/dl to mmol/l, divide by 6.)
Adults: 3.3 - 7.7 mmol/l (20-46 mg/dl)
Infants: 1.3 5.8 mmol/l (8-35 mg/dl)
28
DATE:
Exp No.5 ESTIMATION OF UREA

AIM:

To estimate the level of urea in the given sample by Berthlot method.

PRINCIPLE:
Urease breakdown urea to ammonia and carbon dioxide in alkaline medium, ammonia liberated
from the breakdown of urea reacts with hypochlorite and salicylate to form dicarboxyl. This reaction is
catalyzed by the presence of nitroprusside. The intensity of the color produced is directly proportional to
the concentration of urea present in the sample and is measured photometrically at 600 nm.

CLINICAL SIGNIFICANCE:
Urea is the main waste product of protein breakdown. It is formed in the liver from carbon dioxide
and ammonia, passes into the extracellular fluid, and is excreted almost entirely by the kidneys. The
measurement of urea is an important investigation in diagnosing kidney damage.
Higher level of blood urea than the normal level are usually found with retention of urine due to pre renal
and post renal factors. Dehydration, reduced rate of glomerular filtration and increase catabolism of protein
are the usual pre-renal factors. Renal factors include all sorts of kidney disease and post renal conditions
are enlargement of prostate, stones in the urinary tract, stricture urethra and tumor of the bladder.

REAGENTS REQUIRED:
1. Enzyme Reagent - Dissolve the enzyme reagent in deionized water as per the volume
indicated on vial .
2. Chromogen Reagent
3. Test sample
4. Standard solution (40 mg /dl)

PROCEDURE:
1. Add 1 ml of working reagent in three test labelled as blank , test and standard as per the table .
2. Mix the contents of each tube thoroughly.
3. Place in a boiling water bath for exact 5 minutes.
4. Cool immediately in cold water for 5 minutes.
5. Add 1 ml of chromogen reagent to all test tubes and incubate at 37oC for 5 minutes.
6. Measure the Absorbance of Test and Standard at 600 nm against Blank.

RESULT:

29
CHOLESTEROL ESTIMATION:
OBSERVATION TABLE :

S.NO Particulars B S1 S2 S3 S4 S5 T1 T2

1 Volume of working
standard solution
(ml)

2 Concentration of
working standard
(mg)

3 Serum Sample (ml)

4 Volume of diluted
ferric chloride
solution (ml)

5 Volume of Conc.
H2S04 (ml)

6 Optical Density at
540 nm

30
DATE:
Exp No. 6 ESTIMATION OF CHOLESTEROL BY ZAKS METHOD

AIM:
To estimate the amount of cholesterol in the given serum sample.

PRINCIPLE:
In this method, proteins in serum are precipitated with ferric chloride acetic acid reagent. The
protein free filtrate containing cholesterol is treated with concentrated sulphuric acid. Cholesterol in
presence of sulphuric acid undergoes dehydration to form 3, 5-cholestadiene. This is in turn oxidized and
sulphonated to form red colored cholestapolyene Sulphonic acid in the presence of Fe3+ ions. The
intensity of red color formed is proportional to the amount of cholesterol present in the serum. The color
intensity is measured by using a green filter (540 nm).

CLINICAL SIGNIFICANCE:
Normal serum cholesterol level in young adults is in the range, 150 to 220 mg / dl. It should be
preferably below 200mg/dl. Cholesterol level increases with age.
Increase in serum cholesterol level (Hypercholesterolemia) is seen in :
Hypothroidism
Uncontrolled Diabetes Mellitus
Nephrotic syndrome
Obstructive Jaundice
Decrease serum cholesterol level ( Hypocholesterolemia ) is seen in :
Hyperthroidism
Anemia
Hemolytic Jaundice
Measurement of serum cholesterol level is clinically important, since Hypercholesterolemia is a
causative agent for atherosclerosis and coronary heart disease.

REAGENTS REQUIRED:
1.Stock Standard Solution: Dissolve 100 mg of cholesterol in 100 ml standard measuring flask and
make up to 100 ml with glacial acetic acid (C oncentration =1 mg / ml).
2. Working Standard : Dilute 10 ml of stock solution to 100 ml with ferric chloride - acetic acid reagent
(Concentration = 100 g / ml).
3. Ferric chloride of 0.05% in acetic acid.
4. Glacial acetic acid.

31
CALCULATION:

32
PROCEDURE:
Preparations of protein free filtrate:
Pipette 0.1 ml of serum into a dry test tube and add 9.9 ml of ferric chloride acetic acid reagent.
Mix well by using a glass rod and allow to stand for 10 to 15 minutes. Filter into a dry test tube.
Volume of test sample:
10 ml of protein free filtrate 0.1 ml serum
5 ml of protein free filtrate 0.05 ml serum
Cholesterol Estimation:
In to a series of test tubes (S1,S2,S3,S4,S5), pipette 0.5 ml ,1,1.5,2,2.5 ml of working standard
accordingly. Pipette 1.5 ml of Protein free serum sample into test tube labeled as T. Make up the volume of
all test tubes to 5 ml with FeCl3 solution and 5 ml of FeCl3 serve as the blank.
To all the tubes, add 4 ml of concentrated sulphuric acid. Mix by swirling. Let stand for 20-30
minutes. Read the optical densities by using a green filter (540nm).
Draw a standard calibration curve by taking concentration of Cholesterol on X axis and absorbance
on Y axis.
Calculate the concentration of unknown Cholesterol from the graph.

Normal Value:
Total cholesterol
Desirable < 200 mg/dl
Borderline high 200 239 mg/dl
High > 239 mg/dl

RESULT:

33
OBSERVATION TABLE: Plotting of Standard Graph

Substrate Enzyme Oxaloacetate Distilled DNPH Incubation NaOH O.D. Value

Reagent(ml) Activity Standard water at 505 nm
(ml) (ml)
(ml) (ml)

0.50 (B) 0 0 0.10 0.50 20 min 5.00

0.45 (S1) 24 0.05 0.10 0.50 20 min 5.00

0.40 (S2) 61 0.10 0.10 0.50 20 min 5.00

0.35 (S3) 114 0.15 0.10 0.50 20 min 5.00

0.30 (S4) 190 0.20 0.10 0.50 20 min 5.00

34
DATE:
EXP.No.7.a. ASSAY OF SGOT
AIM:
To estimate SGOT level in the given sample.

PRINCIPLE:
Aspartate aminotransferase (AST) catalyzes the transfer of the amino group from L-aspartate to
ketoglutarate to yield oxaloacetate and L-glutamate. Malate dehydrogenase (MDH) catalyzes the reduction
of oxaloacetate with simultaneous oxidation of NADH+ to NAD. Oxaloacetate so formed is coupled with
3,4 diphenyl hydrazine (2,4 DNPH ) to form corresponding hydrazone, a brown coloured complex in
alkaline medium and this can be measured colorimetrically.
The reaction does not obey Beers Law and hence a calibration curve is plotted using a pyruvate
standard. The activity of SGOT (ASAT) is read off this calibration curve.

L Asparatate Ketoglutarate Oxalaocetate Glutamate

Oxaloacetate 2, 4 DNPH 2, 4 Dinitrophenyl Hydrozone ( Brown coloured complex)

CLINICAL SIGNIFICANCE:
SGOT is an enzyme found mainly in heart muscle, liver cells, skeletal muscle and kidneys. Injury
to theses tissues results in the results of enzyme in blood stream. Elevated levels are found in myocardial
infarction, cardiac operations, Hepatitis, Cirrhosis, acute pancreatitis, acute renal diseases and primary
muscle diseases. Decreased levels may be found in pregnancy, Beri Beri and Diabetic ketoacidosis.
The SGOT test measures the amount of a substance called glutamic-oxaloacetic transaminase
(GOT) in your blood. It is an enzyme found in the liver, muscles (including the heart), and red blood cells.
It is released into the blood when cells that contain it are damaged. Other names for this enzyme are
aspartate aminotranskinase, aspartate transaminase, and AST.

REAGENTS REQUIRED:
Substrate Reagent
DNPH reagent
NaOH reagent (4N)
Pyruvate standard

REAGENTS PREPARATION:
All reagents are ready to use except NaOH reagent (4N) which has to be diluted 1: 10 with distilled
/ deionised water.

35
ENZYME ASSAY

Addition Sequence (B) ml (T) ml

Substrate reagent (L1) 0.50 0.50

Incubate at 37C for 3 minutes

Sample 0.10

Mix well and incubate at 37C for 60 minutes

DNPH reagent (L2) 0.50 0.50

Mix well and allow to stand at RT for 20 minutes

Distilled water 0.10 -

Working NaOH reagent (L3) 5.00 5.00

Optical Density at 505 nm

CALCULATION:
O.D Value =
Enzyme activity =

36
WORKING NaOH REAGENT:
Dilute the sodium Hydroxide to 250ml or for every 1.0 ml of NaOH reagent (4N) add 9 ml of distilled
water. The working sodium Hydroxide reagent is stable at R.T till the expiry mentioned, in a plastic bottle.

SAMPLE MATERIAL:
Serum free from hexolysis SGOT (ASAT) is reported to be in stable in serum for 3 days at 2 8C.

PROCEDURE: Preparation Of Standard Graph

Label the test tube as B,S1,S2,S3andS4.Add Pyruvate standard in the range of
0.05,0.10,0.15,0.20,0.25 ml to B, S1,S2,S3 and S4 respectively. Make up the reaction mixture volume to
0.5 ml with substrate reagent and 0.5 ml of substrate reagent serve as the blank in test tube labelled B. Add
0.1 ml of distilled water to all test tubes. Add 0.5 ml of DNPH reagent to all test tubes. Incubate at room
temperature for 20 minutes. Add 5 ml of NaOH into each of the test tubes. Measure the absorbance at 505
nm against the blank.

ENZYME ASSAY:

Pipette 0.5 ml of substrate reagent into clean dry test tube labeled as Blank (B) and Test (T). Add
0.1 ml of sample into Test tube labelled as T. Mix well and allow standing at room temperature for 30
min.
Add 0.5 ml of DNPH reagent to both test tubes .mix well and allow to stand at room temperature
for 30 minutes. Add 5 ml of working NaOH reagent . Measure the absorbance of the test (T) against Blank
containing 0.1 ml distilled water and read the activity of the test from this calibration curve plotted earlier.
Wavelength / Filter : 505 nm (Hg 546nm) / green
Temperature : 37C & RT
Light Path : 1 cm

NORMAL REFERENCE VALUES:

Serum: 8 40 Units / ml

RESULT:

37
MODEL GRAPH

OBSERVATION TABLE:PREPARATION OF STANDARD GRAPH

Substrate Enzyme Pyruvate Distilled DNPH Incubation NaOH O.D. Value

Reagent(ml) Activity Standard water at 505 nm
(ml) (ml)
(ml) (ml)

0.50 (B) 0 0 0.10 0.50 20 min 2

0.45 (S1) 24 0.05 0.10 0.50 20 min 2

0.40 (S2) 61 0.10 0.10 0.50 20 min 2

0.35 (S3) 114 0.15 0.10 0.50 20 min 2

0.30 (S4) 190 0.20 0.10 0.50 20 min 2

38
DATE:
Exp.No.7.b.ASSAY OF SGPT

AIM:
To estimate level of Blood SGPT (Liver function test)

PRINCIPLE:
SGPT converts - Alanine, - Ketoglutarate to pyruvate and Glutamate. The pyruvate formed
reacts with 2, 4 Dinitrophenyl hydroazine to produce a hydrozone derivative, which is an alkaline medium,
produce a brown coloured complex whose intensity measured. The reaction does not obey Beers law and
hence a calibration curve is read plotted using a pyruvate standard. The activity of SGPT is read off this
calibration curve.
SGPT
L Aspartate Keboglutarate Pyruvate Glutamate
pH 7.4
Alkaline
Oxaloacetate 2,4 DNPH 2,4 Dinictrophenyl Hydrozone
Medium

CLINICAL SIGNIFICANCE:
SGPT is found in a variety of tissues bit is mainly found in lines. Increased levels are found in
nepotistic, cirrhosis, obstructive jaundice and other hepatic diseases. Slight evaluation of the enzymes is
also seen in myocardial infarction.

REAGENTS REQUIRED:
1. Substrate reagent
2. DNPH reagent
3. NaOH reagent
4. Pyruvate Standard

All reagents are ready to use except NaOH reagent (4N) which has to be diluted 1.10 with distilled /
deionised water.

Working NaOH reagent:

Dilute the sodium Hydroxide to 250ml or for essay 1.0 ml of NaOH reagent (4N) add 9 ml of
distilled water. The working sodium Hydroxide reagent is stable at R T till the expiry mentioned in a
plastic bottle.

Sample material:

Serum free from hemolysis SGPT / (AAT) is reported to be stable in serum for 3 days at 2 8C.

39
ENZYME ASSAY

Addition Sequence (B) ml (T) ml

Substrate reagent (L1) 0.50 0.50

Incubate at 37C for 3 minutes

Sample 0.10

Mix well and incubate at 37C for 60 minutes

DNPH reagent (L2) 0.50 0.50

Mix well and allow to stand at RT for 20 minutes

Distilled water 0.10 -

Working NaOH reagent (L3) 5.00 5.00

Optical Density at 505 nm

CALCULATION:
O.D Value =
Enzyme activity =

40
PROCEDURE: PREPARATION OF STANDARD GRAPH
Label the test tube as B,S1,S2,S3andS4.Add Pyruvate standard in the range of
0.05,0.10,0.15,0.20,0.25 ml to B, S1,S2,S3 and S4 respectively. Make up the reaction mixture volume to
0.5 ml with substrate reagent and 0.5 ml of substrate reagent serve as the blank in test tube labelled B. Add
0.1 ml of distilled water to all test tubes. Add 0.5 ml of DNPH reagent to all test tubes. Incubate at room
temperature for 20 minutes. Add 5 ml of NaOH into each of the test tubes. Measure the absorbance at 505
nm against the blank.

ENZYME ASSAY:

Pipette 0.5 ml of substrate reagent into clean dry test tube labeled as Blank (B) and Test (T). Add
0.1 ml of sample into Test tube labelled as T. Mix well and allow standing at room temperature for 30
min.
Add 0.5 ml of DNPH reagent to both test tubes .mix well and allow to stand at room temperature
for 30 minutes. Add 5 ml of working NaOH reagent . Measure the absorbance of the test (T) against Blank
containing 0.1 ml distilled water and read the activity of the test from this calibration curve plotted earlier.
Wavelength / Filter : 505 nm (Hg 546nm) / green
Temperature : 37C & RT
Light Path : 1 cm

NORMAL REFERENCE VALUES:

Serum: 8 40 Units / ml

RESULT:

41
SDS- POLYACRYLAMIDE GEL ELECTROPHORESIS SET UP

42
DATE:

Exp. No.8. SEPERATION OF PROTEINS BY PAGE ELECTROPHORESIS

AIM:

To separate the proteins present in the given sample as charged particles in the presence of
electrical field.

PROCEDURE:

1. Take out the gel solutions from the fridge and leave then at room temperature.

2. Clean the plates after soaking overnight in a detergent wash thoroughly with water and dry in a hot
air oven.

3. Place the notched plate on a paper. Apply vacuum grease and fix three 1.0mm flexi glass spaces
strips on both the sides and at the bottom. Place the regular plate, with 6 clips.

4. Mix the separating gel solutions gently omitting SDS and APS. Degas the solution.

5. Now add SDS and take ~0.1 ml of the separating gel solution in a small test tube and 20 ml APS.
Leave it for 5 10 min. Check whether it has polymerized. If it has polymerized proceed to the
next step.

6. Take 0.1ml of the separating gel solution and add 20 ml APS and add quickly through the inner
edges of the plate. This will polymer immediately and seal the bottom.

7. Allow the gel to stand for 30 min to 60 min to polymerize. A clear butanol gel interphase will be
visible.

8. Once the gel is polymerized, pour off the top layer by tilting the gel, wash gel surface gently with
distilled water.

9. Mix the gel solutions gently for stacking gel as given above.

10. Rinse the top of the gel with ~ 0.1 ml of stacking gel solution and pour off.

11. Fill the top of the gel with stacking solution

12. Insert the comb gently between the plates. Take care not to trap any air bubbles.

13. Allow the gel to stand for 30 mins to polymerize.

14. Remove the bottom clips and bottom spaces

15. Remove the comb vertically upwards.

16. Apply vacuum grease and fix the glass plates with the gel tank tightly with clips on both the sides.

17. Fill the upper and lower chambers with running buffer. Connect leads to DC power supply.

43
44
 18. Prepare sample solutions by mixing in 2x sample buffer and placing the tube for 5 mins in a
boiling.

19. Using a syringe with bent needle, remove air bubbles under the gel between the glass plates in the
bottom tank.

20. Electrophoresis the gel at 80 volts until the tracking dye has reached ~ 0.5 cm from the bottom of
the gel.

21. Turn off power supply, disconnect the leads and remove the glass plates from the tank.

22. Gently slide the gel into a tray containing dye solution.

23. Stain the gel for 2 3 hours or overnight at room temperature.

24. Pipette out the staining solution and replace destaining solution 2 3 lts.

RESULT:

45
 THIN LAYER CHROMATOGRAPHY CHAMBER

CALCULATION:

Rf = Distance moved by solute

---------------------------------
Distance moved by solvent

46
DATE:

Exp.No. 9 SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY

AIM:

To separate amino acids by thin layer chromatography.

PRINCIPLE:

Chromatography is a method by which a mixture of substances in smaller quantities can be

separated both qualitatively and quantitatively. In chromatography there are two phases-the stationary
phase and other mobile phase. When the mobile phase moves along stationary phase, separation of
substances takes place. In thin layer chromatography, the thin layer of gel functions as an inert supporting
material. When the mobile phase moves along the gel solvent, as the partition coefficient differ for
different sugars, the rate of flow differs and therefore separation occurs.

MATERIALS REQUIRED:

1. Silica gel G
2. Microscopic slides
3. n-butanol
4. Acetic acid
5. Spraying reagent (0.3% solution of Ninhydrin in butanol containing 3 ml acetic acid)
6.Amino acids
Solvent system:

1. n Butanol / Acid / water (8:2:2)

2. 96% Ethanol / Water (7:3)
3. Chloroform, methanl, 17% NH4OH

Solvent Reagent:

0.1% solution of ninhydrin in acetone, heat for ~ 10 min o\in a 100C over Elution: 5 ml of 80%
Ethanol or methanol.

47
Rf Values of Some Amino Acids

AminoAcid Rf value
- Alanine 0.39
Arginine 0.19
Cysteine 0.17
Glycine 0.33
Aspartic Acid 0.33
Glutamic Acid 0.37
Lysine 0.18
Methionine 0.51
Valine 0.56
Serine 0.31

48
PROCEDURE:

Thin layer Plate Preparation:

The stationary phase is prepared as a slurry with water or buffer at 1 : 2 and applied to a glass plate
or in an inert plastic or aluminum sheet, as a thin uniform layer by means of a spreader such as a glass rod
or pipette or using a TLC applicator. Calcium sulphate CaSO4.1/2H2O is incorporated to the absorbent as a
binder, as it facilitates the adhesion of the absorbent of the plate. After application of the absorbent, the
plates are air-died for 10 15 min and then ones-died for 10 -15 min at 100C - 110C. This process is
known as activation of the absorbent.

Sample Application:

Draw a line lightly with a pencil. About 1.5 2.0 cm from the bottom , place a scale at the bottom
and spot a distance ~1.5 cm. Note the order. The samples are spotted using a capillary tube at ~1.5cm
distance between them. For preparative TLC, the sample is applied as a band across the layer rather than
a spot.

Plate development:

The chromatographic tank is filled with the developing solvent to a depth of ~ 1.5cm and
equilibrated for about 5 hours. The capillary action causes the solvent to ascend as in paper
chromatography and the separation of compounds take place. As the solvent front reaches about 1 2 cm
from the top of the plate, the plate is removed, solvent front is marked with a pencil immediately and
allowed to air dry placing the plate upside down.

For amino acid the plate can be sprayed with 0.1% ninhydrin in acetone incubated at 100C for 5
10 min.

RESULT:

49
50
DATE:

Exp. No. 10. SEPARATION OF DNA BY AGAROSE GEL ELECTROPHORESIS

AIM:

To separate the DNA present in the given sample by Agarose Gel Electrophoresis.

THEORY:

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying
sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed
genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits .
During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore
sizes determine a gel's molecular sieving properties.

The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption
of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only
provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded
into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA)
molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to
the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are
separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional
to the log of its molecular weight .

The rate of migration of a DNA molecule through a gel is determined by the following:

1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation; 4) voltage

applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer.

PROCEDURE:

PREPARATION OF THE GEL

Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared
using aw/v percentage solution. The concentration of agarose in a gel will depend on the sizes of
the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of
the buffer should not be greater than 1/3 of the capacity of the flask.
Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running
buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM
EDTA).

51
52
 Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but
can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents
to mix well. Repeat until the agarose has completely dissolved.
Add ethidium bromide (EtBr) to a concentration of 0.5 g/ml. Alternatively, the gel may also be
stained after electrophoresis in running buffer containing 0.5 g/ml EtBr for 15-30 min, followed
by destaining in running buffer for an equal length of time.
Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations.
Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the
staining of DNA are available; however EtBr remains the most popular one due to its sensitivity
and cost.
Allow the agarose to cool either on the benchtop or by incubation in a 65 C water bath Failure
to do so will warp the gel tray.
Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a
gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.
Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature.
Remove the comb and place the gel in the gel box Alternatively, the gel can also be wrapped in
plastic wrap and stored at 4 C until use

SETTING UP OF GEL APPARATUS AND SEPARATION OF DNA FRAGMENTS

Add loading dye to the DNA samples to be separated . Gel loading dye is typically made at 6X
concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps
to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.
Program the power supply to desired voltage (1-5V/cm between electrodes).
Add enough running buffer to cover the surface of the gel. It is important to use the same
running buffer as the one used to prepare the gel.
Attach the leads of the gel box to the power supply. Turn on the power supply and verify that
both gel box and power supply are working.
Remove the lid. Slowly and carefully load the DNA sample(s) into the gel . An appropriate DNA
size marker should always be loaded along with experimental samples.
Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the
anode (red leads). Double check that the electrodes are plugged into the correct slots in the
power supply.
Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

53
54
OBSERVING SEPARATED DNA FRAGMENTS

When electrophoresis has completed, turn off the power supply and remove the lid of the gel
box.
Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel
tray on paper towels to absorb any extra running buffer.
Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done
using a gel documentation system. DNA bands should show up as orange fluorescent bands.
Take a picture of the gel.
Properly dispose of the gel and running buffer per institution regulations.

RESULT:

55
56
DATE:

Exp.No.11.a.ERYTHROCYTE SEDIMENTATION RATE (WINTROBE'S METHOD)

AIM:

To calculate the ESR Erythrocyte sedimentation of a given blood sample.

PRINCIPLE:

Erythrocyte Sedimentation Rate (ESR, Sed Rate, and Sedimentation Rate) is a laboratory blood test
that indirectly measures the degree of inflammation in the body. It measures the rate of settling or
sedimentation of red blood cells (erythrocytes) in a tall, thin tube of blood collected from a patient.

Proteins produced during inflammation cause erythrocytes to move close together and stack up in a
group. When red blood cells are in group, they become heavier (denser) so they settle faster. The further
erythrocytes settle and the faster they fall, the higher is the value of ESR. ESR blood test result is reported
as how many millimeters of plasma (clear, yellowish fluid component of the blood) are present at the top
of the thin tube in one hour.

CLINICAL SIGNIFICANCE:

ESR is an easy inexpensive and non specific test, which helps in diagnosis as well as prognosis.
It is non specific because it is cannot indicate the exact location or cause of disease. But it helps to
confirm the diagnosis. ESR increase in diseases like tuberculosis, All type of anemia, except the sickle cell
anemic, malignant tumour matid arthritics, Rheumatic fever, Liver diseases, polycythemia, Extreme
duekocytosis, ESR decreases in the cell such as allergic conditions, sickle cell anemia, peptone shock.

THEORY :

ESR blood test helps in the diagnosis of conditions associated with acute and chronic inflammation
such as infections, cancers, and autoimmune diseases. Although ESR blood test can tell that there is
inflammation in the body, it cannot tell what condition or disease is causing the inflammation. Doctors use
ESR blood test result in conjunction with other clinical findings, laboratory test results, and the patients
health history in diagnosing the disease of a patient.

There are many conditions that may cause inflammation and increase ESR: rheumatoid arthritis, lupus,
anemia, endocarditis, kidney diseases, osteomyelitis, tuberculosis, syphilis, thyroid disease, rheumatic
fever, systemic body infection, giant cell arteritis, pregnancy, multiple myeloma, polymyalgia, vasculitis,
and other inflammatory diseases.

There are also conditions that result in lower-than-normal ESR: sickle cell anemia, polycythemia,
blood hyperviscosity, low plasma protein due to certain diseases, decreased blood fibrinogen levels
(hypofibrinogenemia), and congestive health failure

57
58
MATERIALS REQUIRED:

Wintrobes tube

Anticoagulant : EDTA

Wintrobes Sedimentation Rack

PROCEDURE:

About one ml of blood is mixed with anticoagulant EDTA. The blood is loaded into the Wintrobe
tube without any bubbles up to 0mark and the tube is placed on the Wintrobes stand. Leave for one hour
without disturbing.RBC settle down leaving clear solution above. Height of clear plasma at the top is noted
after one hour and is expressed as ESR (erythrocyte sedimentation rate).It is also called sedimentation rate
(or) Bienacki reaction.

NORMAL RATE:

For men: 3.7- 6.5 mm/hr

For women: 9.6 -15 mm/hr

The rate increases in all acute general infections. It also increases in old age and during pregnancy and
is low in children.

RESULT:

59
DETERMINATION OF HEMATOCRIT

60
DATE:

Exp.No.11.b. PACKED CELL VOLUME

AIM:

To estimate the packed cell volume of the given blood sample.

PRINCIPLE:

When anti coagulated blood is centrifuged , the RBCs which are heavier then the WBCs and platelets
settle at the bottom .This red blood cells column is called hematocrit.

CLINICAL SIGNIFICANCE:

Determination of PCV helps in diagnosis and treatment of anemia, polyeythemia, dehydration and
recovery blood transfusion. It also increase in dengue fever. Decrease in pregnancy, criophsis of liver.

THEORY:

Haemoglobin is a red pigment present in all walls of RBC . It is a respiratory filament that
combines with O2 to form oxyhaemoglobin, it carries oxygen to the tissues. Decreased concentration
below normal value leads to anemia. Hemoglobin vales are lower in women and children, than in men. The
value also gets decreased during pregnancy. Increased hemoglobin concentration can occur due to
thalassemia.

Hematocrit refers to the percentage of total blood cells. The hematocrit value may also be very high
(up to 65%) . Such blood becomes very viscous and person may die due to multiple plugging of peripheral
vascular blood vessels.

PROCEDURE:

Fill the wintrobes haematocrit tube (rinsed with potassium oxaloacetate ) upto 100 mark. Wintrobe
tube is a small graduated tube of uniform bore. Centrifuge the tube for 30 minutes at 3000 rpm till the
packing is complete. The RBC packed at the bottom is the packed cell volume and the clear plasma
remains above this. Its between the RBCs and the plasma, there is a white buffy coat, which is
informed by white blood cells and the platelets. Read the packed cell volume directly from the
graduated given on the tube & express the result in percentage.

NORMAL VALUE:

Male : 41 50 % (Average 47 %)

Female: 36- 44 % (Average 40 %)

RESULT:

61
62
DATE:

Exp. No.11.c DETERMINATION OF BLOOD INDICES (MCV, MCH, MCHC)

AIM:

To determine blood indices like MCV Mean Corpuscular Volume, MCH Mean Corpuscular
Hemoglobin, MCHC Mean Corpuscular Hemoglobin Concentration.

CLINICAL SIGNIFICANCE:

Blood indices are simply meant for erythrocytes. The number shape, volume and the colour of the
RBC indicate the quality of blood. So, these features are named as blood indices.

IMPORTANCE OF BLOOD INDICES:

Blood indices have got diagnostic value in determining the type of anemia.

DIFFERENT BLOOD INDICES:

Following are the various blood indices.

1. Mean Corpuscular Volume

2. Mean Corpuscular Hemoglobin
3. Mean Corpuscular Hemoglobin Concentration
4. Colour Index

MEAN CORPUSCULAR VOLUME (MCV):

MCV is the average value of a single RBC and it is expressed in cubic microns. The normal MCV is
90 cu mm. When MCV increases, the cell is known as macrocyte and when it decreases, the cell is called
microcytic. MCV is mone in pernicious anemia and megaloblastic anemia in which the RBCs are
macrocytic in nature. MCV is less in microcytic anemia.

For example,

PCV-36%

RBC- 4.5 million cells/cubic litre

MCV = PCV/RBC*10

= 36/4.5*10 = 80 cu. Micron

NORMAL VALUE: 78 90 cu .micron

If MCV is less than 78 then it is micro-cytic anemia. If the value is more than 94, it is macrocytic
anemia

63
CALCULATION:

Mean Corpuscle Volume:

Mean Corpuscle Hemoglobin:

Mean Corpuscular Hemoglobin Concentration:

64
MEAN CORPUSCULAR HAEMOGLOBIN (MCH):

It is the quantity or amount of hemoglobin present in one RBC. It is expressed in micro gram or
pictogram. The normal value of MCH is 30 pg. It decreases or remains normal inprenicious anemia and
megaloblastic anemia, in which RBCs are macrocytic and normochromic or hypochromic. It decreases in
hypochromic anemia when MCH is norma, it is called normochromic state.

For example,

Hb = 12.5 g/dl

RBC = 4.8 million cells/ cubic litre

MCH = Hb/RBC*10

= 12.5/4.8 * 10

= 25 Pg

NORMAL VALUE : 27-32 pg

A figure less than 27 picogram indicates hyperchronic anaemia

MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION (MCHC):

It is an expression of the average the concentration per unit volume of packed red cell. This is the
concentration of hemoglobin in one RBC. It is the amount of hemoglobin expressed in relation to the
volume of one RBC. So, the unit of expression is percent. This is the most important absolute value in the
diagnosis of anemic. The normal value of MCHC is 30%. It decreases in iron deficiency anemic in which,
RBCs are microcytic and hypochromic. It is expressed in g/dl or in %

For example,

Hb = 12.5g/dl, PCV = 44 %

MCHC = Hb/PCV * 100 = 12.5/44 * 100= 28.57 %

NORMAL VALUES:

MCV Mean Corpuscular Volume = 78 to 90 cu

MCH Mean corpuscular Hemoglobin = 27 to 32 pg

MCHC Mean Corpuscular Hemoglobin Concentration = 30 to 38%

RESULT:

65
HAEMOGLOBIN STATUS UNDER DIFFERENT PATHOLOGIC CONDITIONS

HAEMOGLOBINOMETER

66
DATE:

Exp.No. 11.d ESTIMATION OF HAEMOGLOBIN CONTENT BY SAHLIS METHOD

AIM:

To estimate the amount of hemoglobin present in the blood.

PRINCIPLE:

When blood is added to 0.1N HCl, hemoglobin is converted into brown coloured acid haematin. The
settling colour after dilution is compared with standard reference of hemoglobinometer.

CLINICAL SIGNIFICANCE:

A decrease in hemoglobin below the normal range is an indication of anemia. Hemoglobin level
decreases in pregnancy, blood modulation polycythemia congenital heart diseases.

MATERIALS REQUIRED:

Anticoagulated blood, Sahlis Haemoglobinometer, calibrated unit for haemoglobin measurement

Sahlis Pipette , 0.1N HCl, distilled water, pasture pipette.

HAEMOGLOBINOMETER:

It consists of graduated tube called a dilutant tube. The set has two vertical tubes with standard
brown color and in between is a graduated tube in which blood sample is taken.

PROCEDURE:

Take 0.1N HCl is added up to the lowest 20 mark on the percentage side of the graduated tube.
Draw 20 ml of the blood in the Sahlis pipette and wipe the excess blood with the help of dry cotton. Put
this blood in the graduated tube and stir with the stirrer provided and match the color developed with the
given brown color on either side. Add more distilled water drop wise and mix till the colour matches the
standard given colour. Remove the graduated tube and read the upper meniscus to get haemoglobin
percentage in gm/dl.

NORMAL VALUE:

Newborns: 17-22 gm/dl, Children: 11-13 gm/dl

Adult males: 14-18 gm/dl , Adult women: 12-16 gm/dl

Men after middle age: 12.4-14.9 gm/dl

Women after middle age: 11.7-13.8 gm/dl

RESULT:

67
NEUBAUER'S COUNTING CHAMBER:

SQUARES IN COUNTING CHAMBER

68
DATE:

Exp 11 e. TOTAL ERYTHROCYTE (R.B.C.) COUNT

AIM:

To estimate the total amount of erythrocyte (RBC) given in the blood sample.

CLINICAL SIGNIFICANCE:

RBC count has great physiological significance. It varies under different conditions. Decrease in
total erythrocyte count is seen in haemoconcentration due to burns, cholera etc. Increased count of
erythrocyte is observed in polycythemia.

REQUIREMENTS:

Binocular microscope,. Neubauer counting chamber, RBC diluting fluid, sahlis pipette.

NEUBAUER'S COUNTING CHAMBER:

The central part of the counting chamber has an upper and lower counting chamber. In each
chamber there are four large squares one in each corner with 1 mm 3 area and there are 16 small squares in
each corner square. The center 1 mm3 area is divided into 25 (5x5) squares separated by triple lines and
each square is divided into 16 (4x4) small squares .

RBC DILUTING FLUID PREPARATION:

Trisodium citrate 3 gm

Formaline 1 ml

Distillled water 100 ml

PROCEDURE:

The RBC pipette ( Pipette with red ball ) has three graduations 0.5 and 1 on the stem and a third
mark 101 just above the bulb. Fill the RBC pipette exactly with capillary blood upto 0.5 mark by holding
the pipette horizontally. Fill rest of the pipette with the diluting fluid upto the mark 101. The bulb is
constructed such that it contains fluid exactly 100 times the volume contained up to mark 1, so the blood
dilution is 100 times. If the blood is taken up to mark 0.5 the dilution is 200 times. Mix well by rotating the
pipette , and take 1-2 drops on the Neubauer chamber and put the cover slip .

69
CALCULATION:

No. of cells counted dilution

Number of RBC / ml
Area counted depth of fluid
Dilution = 200

Area counted = 2.5x0.04=0.2

Depth of fluid = 0.1 mm

Dilution 200
Factor 10000
Volume 0 .2 0 .1

RBC / cu mm of blood = No. of Cell x 10000

First cell -

Second cell -

Third cell -

Fourth cell -

Fifth cell -

TOTAL=

Mean =

RBC / cu mm of blood =

70
COUNTING THE SQUARES - RBC COUNTING

Place the counting chamber on the stage of microscope. Switch to low power objective. Adjust the light
and rotate the large square in the centre with 25 small square. Count the cells in each corner and one
central square and record . If the cells are on the marginal lines , count the cells in the left and lower
margins and discard the ones on the right and upper marginal line. In this way the cells are counted in 16x5
small squares.

NORMAL VALUES:

Male - 4.56 million cells / cu mm

Female - 4.45 million cells / cu mm

RESULT:

71
72
AUGMENTED EXERCISES

73
DETERMINATION OF BLOOD GROUPS

74
DATE:

Exp 12. BLOOD GROUPING

AIM:

To identify blood groups in given sample using agglutinogen.

IDENTIFICATION OF BLOOD GROUP:

Blood grouping is carried out on the basis of the agglutinogens which are present in the RBC.
There are four blood groups based on the presence or absence of agglutinogens in RBCs. These are A, B,
AB, and O blood group. There are two primary agglutinogens A and B and two corresponding agglutinins
and present on the surface of the RBC. The RBCs may contain only A or only B, i.e blood group A or
B, or both A and B i.e. blood group AB or no agglutinogen A and antigen B or blood group O. Hence
blood group AB is universal recipient and blood group O is universal donor.

A, B, O blood grouping can be performed in two ways:

1. Forward Grouping

2. Reverse Grouping

FORWARD GROUPING: In forward grouping antigenic character of RBC are determined by reacting
with corresponding antibody.

REVERSE GROUPING: The character of the antibody is determined with expected antibody that
should be able to tally with it. In the blood group established by forward grouping, red cells are used in
forward grouping and serum is used for reverse grouping.

MATERIALS REQURIED:

Glass slide, spirit, cotton, antiSera A, antisera B, disposable needle.

PROCEDURE:

Prick the left finger with a pricker and take drop of blood on two seperate spots on a clean dry slide
Put antisera A on the first spot and antisera B on the second spot. Mix well and see the formation of
precipitate. Interpret the result as given below:

If precipitate react with A- the blood group is A

If precipitate react with B- the blood group is B

If precipitate react with A&B- the blood group is AB

If precipitate react with none - the blood group is O

RESULT:

75
76
DATE:

Exp 12. DIFFERENTIAL COUNT

AIM:

To prepare human blood smear and differentiate leucocytes

PRINCIPLE:

Three major steps involved in different count are preparation of smear, staining of smear,
microscopic observation.

The smear is made with a drop of blood directly from the skin puncher, which gives the true picture
of blood morphology staining is done with play chromatic strain, which includes Methylene blue and Eosin
Leishmans stain. The polychromatic stains include multiple colours when applying to the cells. The stain
dissolves in methanol and buffer from pH 7 to 7.2. Methanol acts as a fixing agent and also occurs as a
solvent.

Following the staining basic compounds of white cells are stained by acoustic dye and are
described as eosinophils or acidophilic where the acetic compound of blood (nucleus and nucleic acid).
Take blue to purple shade by the basic dyes and are called basophilic. The neural components present in
the blood cells are probably stained by both of the dyes.

MATERIALS REQUIRED:

Leishman's stain, Binocular microscope with light, Glass slides, Cover slip, Pricker.

PROCEDURE:

Preparation of smear:

With sterilized pricker, prick the ring finger and take blood on a clean dry glass slide. With another
spreader slide kept at 45oC , draw the blood backwards to make a thin film.

Fixing and staining:

Dry the blood film and put 2-3 drops of Leishman's stain. Methanol present in the stain fixes the
smear. Keep the slide on the staining rack with the blood smear facing up. After 5-7 minutes, drain off the
stain by tilting the slide and wash under slow running tap water for 5-10 minutes. Now dry the slide
slightly and mount the slide in Glycerine without any air bubbles.

Observation of blood smear:

Observe different types of cells under the microscope , draw the cells observed and identify them.

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NORMAL VALUES:

Neutrophils 50 70%

Lymphocytes 20 40%

Eosinophils 1 3%

Monocytic 15%

Basophilic 1%

RESULT:

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