R. D. & S.H. National College and S. W. A.

Science College
Bandra (W), Mumbai – 400 050

Department of Biotechnology
Laboratory Journal
M.Sc. Pt. I
Semester I
 R. D. & S.H. National College and
S. W. A. Science College

Department of Biotechnology
Laboratory Journal
M.Sc. Pt. I
Semester I

NAME: AISHWARYA BALIRAM DIWALE

Roll No: BT20003
 Department of Biotechnology
R.D. & S.H. National College and S.W.A Science College,
Bandra (W), Mumbai – 400 050.

CERTIFICATE

This is to certify that Ms. AISHWARYA BALIRAM DIWALE

of class M.Sc.-I, Department of Biotechnology, R.D. & S.H. National College
and S.W.A. Science College, has satisfactorily completed the practical
course for Semester-I of M.Sc. Biotechnology prescribed by University of
Mumbai during the academic year 2020- 2021 The work entered in journal is
the bonafide work of the student as carried out in Biotechnology
Laboratory of the College.

Signature Faculty Signature

Incharge Head
Department of Biotechnology

Date:

Department Stamp
 INDEX
Practical I PSBT 101 & PSBT 104
Sr. Experiment Title Pg. Date Signature
No. Nos.
1. To prepare Acetate and Phosphate buffers 6 – 11 23/2/21
using the Henderson-Hassel Bach equation
2. To determine an unknown protein 12– 13 23/2/21
concentration by plotting a standard graph of
BSA using UV-Vis Spectrophotometer and
validating the Beer- Lambert‘s Law
3. Identification of sugars in fruit juices using 14 – 15 23/2/21
thin layer chromatography
4. Protein gel staining techniques: silver 16 – 19 27/2/21
staining, Activity staining: LDH,
glycoprotein staining
5. Viscosity studies of proteins 20 – 21 27/2/21
6. Isolation of starch from potato and its 22 – 24 27/2/21
estimation by anthrone method
7. The isolation and assay of glycogen from 25 – 27 1/3/21
liver and skeletal muscles of bird/mammal
8. Antimicrobial sensitivity test and 28 – 29 1/3/21
demonstration of drug resistance
9. Photo album of chromosomal abnormalities 30 1/3/21
in normal and disease condition, numerical
detected by using different probes –
centromeric, locus specific, telomeric
Structural - Translocations and fusion genes
Detection of inversions and interstitial
deletions by SKY CGH for a disease or
cancer
 Practical II PSBT 102 & PSBT 103
Sr. Experiment Title Pg. Date Signature
No. Nos.
1. Preparation of TAB and sterility testing 31 - 37 22/2/21
2. Perform serum electrophoresis (horizontal) 38 - 41 22/2/21
3. Latex bead agglutination / precipitation test 42 - 43 22/2/21
for detection of rheumatoid factor (RF)
4. Separation of lymphocytes on Ficol 44 - 46 24/2/21
Histopaque and viability count
5. In-vitro demonstration of phagocytosis and 47 - 49 24/2/21
calculating phagocytic index
6. Cell permeability testing- osmotic fragility 50 - 53 24/2/21
7. Isolation of cell organelle by differential 54 - 56 26/2/21
centrifugation techniques from plant / animal
sources
8. Isolation of mitochondrial DNA 57 - 59 26/2/21
9. Isolation of chloroplast DNA 60 - 62 26/2/21
10. Cell death /apoptosis studies using flow- 63 - 64 26/2/21
cytometry demonstration
 1. PREPARATION OF ACETATE AND PHOSPHATE BUFFERS USING THE
HENDERSON-HASSEL BACH EQUATION

AIM: To prepare Acetate and Phosphate buffers using the Henderson-Hassel Bach equation

KEY PRINCIPLE:
A buffer is a solution that can resist pH change upon the addition of an acidic or basic
components. It is able to neutralize small amounts of added acid or base, thus maintaining the
pH of the solution relatively stable. Buffers are significant in many areas of chemistry. When
its comes to buffer solution one of the most common equation is the Handerson-Hasselbalch
approximation. To prepare a buffer that would tend to have and maintain a given pH value, a
solution of weak acid or base and a solution of salt of the acid or base is needed. Given the
value of Ka of the acid, and the [H+] required in an acid buffer the formula below can be used
to determine the [HA]/[A] ratio in the buffer.
K = [H+] [A-]
a

[HA]

Buffer Capacity = No. of moles of OH or H O added

-
3
+

(pH changes) (Volume of buffer in litre)

By mixing appropriate volumes of the acid and salt solutions the concentration ratio can easily
be obtained and thus the buffer can be made.

The Henderson-Hasselbalch equation can be written as: pH = pKa + log 10 ([A – ]/[HA])Where
[A – ] denotes the molar concentration of the conjugate base (of the acid) and [HA] denotes the
molar concentration of the weak acid
REQUIRMENTS:

(I) Phosphate buffer:

Potassium Phosphate

Phosphonic Acid

(II) Acetate buffer:

Sodium Acetate and Acetic acid
(III) Miscellaneous: pH meter, Balance, Glassware.

CALCULATIONS:
Problem 1: Prepare 50ml of 0.5M Phosphate buffer of pH 6.4 and pKa 6.1 using 0.1M
Potassium Phosphate and 0.1M Phosphonic Acid.

Solution:- Step 1: Use Henderson- Hasselbach equation to find the ratio of A (basic) and HA
- +

(acidic) component

pH = pK + log [A ]
a
-

[HA ] +

6.4 = 6.1 + log [A ] -

[HA ] +

0.3 = log [A ] -

[HA ] +

Taking log on both sides,

1.99 = [A ]
-
……….(1)

[HA ] +
 Rewriting equation (1) we get,

1.99 = [A ] -

1 [HA ] +

Step 2: Calculate the decimal fraction of each buffer component,

Total difference of buffer = 1.99 + 1= 2.99

Decimal fraction of [A ] = 1.99/2.99 = 0.66

-

Decimal fraction of [HA ] = 1/2.99 = 0.33

+

Step 3: Find the molarity for each component of the buffer by multiplying molarity of buffer to
the decimal function of each component

M = 0.66 x 0.5 = 0.33

A

M = 0.33 x 0.5 = 0.16

HA

Step 4: Calculate the moles of each component in the buffer using the formula-

Moles = Molarity x Litres of buffer

Moles = 0.33 x 0.05 = 0.016

A

Moles = 0.16 x 0.05 = 0.008

AH

Step 5: Calculate the volume of each stock solution required to make the buffer by using the
formula-

Litres of Stock = Moles of buffer component

Molarity of stock

Litres of Stock = 0.016/ 0.5 = 0.032 l =32 ml

A

Litres of Stock = 0.008/ 0.5 = 0.016 = 16 ml

HA
 To prepare 50 ml of Phosphate buffer, we require 32 ml of 0.5 M Potassium phosphate and 16 ml
of 0.5 M Phosphoric acid which sums up to 48ml. To make up the volume of the buffer to 50ml,
2 ml of Distilled water is to be added.

Problem 2: Prepare 100ml of 0.03M Acetate buffer of pH 3.8 and 4.76, the concentration of
Sodium Acetate and Acetic Acid is 0.3M.
Solution- Step 1: Use Henderson- Hassel Bach equation to find the ratio of A (basic) and HA
- +

(acidic) component,
pH = pK + log [A ]
a
-

[HA ] +

3.8 = 4.76 + log [A ] -

[HA ] +

-0.96 = log [A ] -

[HA ] +

Taking log on both sides,

0.10 = [A ]-
……….(1)

[HA ] +

Rewriting equation (1) we get,

0.10 = [A ] -

1 [HA ] +

Step 2: Calculate the decimal fraction of each buffer component,

Total difference of buffer = 0.10 + 1= 1.10

Decimal fraction of [A ] = 0.10/1.10 = 0.09

-

Decimal fraction of [HA ] = 1/1.10 = 0.90

+
Step 3: Find the molarity for each component of the buffer by multiplying molarity of buffer to
the decimal function of each component

M = 0.09 x 0.03 = 0.0027

A

M = 0.90 x 0.03 = 0.027

HA

Step 4: Calculate the moles of each component in the buffer using the formula-

Moles = Molarity x Litres of buffer

Moles = 0.0027 x 0.1 = 0.00027

A

Moles = 0.027 x 0.1 = 0.0027

AH

Step 5: Calculate the volume of each stock solution required to make the buffer by using the
formula-

Litres of Stock = Moles of buffer component

Molarity of stock

Litres of Stock = 0.00027/ 0.3 = 0.0009 =0.9 ml

A

Litres of Stock = 0.0027/ 0.3 = 0.009 = 9ml

HA

To prepare 100 ml of 0.03 M Acetate buffer, we require 0.9 ml of 0.3 M Sodium acetate and 9
ml of 0.3 M Acetic acid which sums up to 9.9 ml. To make up the volume of the buffer to 100ml,
90.9 ml of Distilled water is to be added.
RESULT:

Problem 1:- To prepare 50 ml of Phosphate buffer of pH 6.4 and pK 6.1 using 0.5 M Potassium
a

phosphate and 0.5 M Phosphoric acid; we require 32 ml of Potassium phosphate and 16 ml of

Phosphoric acid which sums up to 48ml. To make up the volume of the buffer to 50ml, 2 ml of
Distilled water is to be added.

Problem 2:- To prepare 100ml of 0.03 M Acetate buffer of pH 3.8 and pK 4.76 using 0.3 M
a

Sodium acetate and 0.3 M Acetic acid; we require 0.9 ml of Sodium acetate and 9 ml of Acetic
acid which sums up to 9.9 ml. To make up the volume of the buffer to 100ml, 90.9 ml of
Distilled water is to be added.

CONCLUSION:

Phosphate and Acetate buffers of desired concentrations were prepared using the Henderson-
Hassel Bach equation.
2. DETERMINATION OF UNKNOWN PROTEIN CONCENTRATION BY PLOTTING
A STANDARD GRAPH OF BSA USING UV-VIS SPECTROPHOTOMETER AND
VALIDATING THE BEER- LAMBERT’S LAW

AIM: To determine an unknown protein concentration by plotting a standard graph of BSA

Using UV-Vis Spectrophotometer and validating the Beer- Lambert‘s Law.

KEY PRINCIPLE:
Spectroscopy is a technique that measures the interaction of molecules with electromagnetic
radiation. Quantitation of the amount of protein in a solution is possible in a simple
spectrometer. Proteins usually show absorption maxima between 275 and 280nm. Absorption
of radiation in the near UV by proteins depends on the Tyr and Trp content. The advantages of
this method are that it is simple, and the sample is recoverable. The method has some
disadvantages, including interference from other chromophores, and the specific absorption
value for a given protein must be determined

REQUIREMENTS: Bovine serum albumin, PBS, spectrophotometer, protein sample

KEY OBSERVATION:
Conc. Of stock Absorbance at
(µg/ml) 280nm

10 0.15
20 0.25
30 0.36
40 0.46
50 0.60
60 0.72
70 0.78
80 0.83
90 0.91
100 1.06
Uk 0.33
RESULTS:

The concentration of the unknown sample is calculated by the equation: y = mx + c

By determining the graph, substituting the value of y, m and c in the above equation.
y = 0.0098x + 0.0707
0.33 = 0.0098x + 0.0707
0.33 - 0.0707 = 0.0098x
0.2593 = 0.0098x
0.2593 / 0.0098 = x
x = 26.45
The unknown concentration “x” is 26.45 µg/ml.
CONCLUSION:
Determination of an unknown protein concentration was successfully carried out by plotting a
standard graph of BSA Using UV-Vis Spectrophotometer. The unknown concentration was
found out to be 26.45 µg/ml by graphical representation.
 3. IDENTIFICATION OF SUGARS IN FRUIT JUICES USING THIN LAYER
CHROMATOGRAPHY

AIM: To identify sugars in fruit juices by Thin-Layer Chromatography

KEY PRINCIPLE:
Thin layer chromatography (T.L.C.) is already accepted as a laboratory tool for routine work. Its
low cost, ease, and rapidity along with its capacity for separating and identifying small quantities
of compound mixtures make the technique a prime tool for research as well. Thin-layer
chromatography (TLC) depends on the separation principle. The separation relies on the relative
affinity of compounds towards both the phases. The compounds in the mobile phase move over
the surface of the stationary phase. The movement occurs in such a way that the compounds
which have a higher affinity to the stationary phase move slowly while the other compounds
travel fast. Therefore, the separation of the mixture is attained. On completion of the separation
process, the individual components from the mixture appear as spots at respective levels on the
plates. Their character and nature are identified by suitable detection techniques. The objective
of this investigation was to adapt a method for separation, identification, and approximation of
different sugars in beet processing liquors, thick juice from storage, and beet storage samples.

REQUIREMENTS:

TLC plates, Solvent system which consists of a mixture of chloroform, acetic acid, and water
(3:3.5:0.5) by volume, respectively, Spraying agent made from 1 gram diphenylamine and 1 ml
of aniline in 100 ml acetone. This mixture is further mixed with 85% orthophosphoric acid prior
to use (10 : 1 v/v, respectively).

KEY OBSERVATION:
RESULT:
Identification of sugars in fruit juices was successfully carried out using thin layer
chromatography. In the sugar mixture 1 on the 2nd well 5 bands was observed on the TLC plate,
whereas in the sugar mixture 2 on the 4th well 4 bands was observed. In the 2nd well, we observed
5 bands which lie on the same plane as the other sugars indicating that the sugar mixture 1 have
all of the five sugar components in it. In the 4th well, we observe 4 bands, out of which 2 bands
match with glucose and saccharose respectively. Whereas the other 2 bands in the 4 th well don’t
any match with any of the sugars, meaning they would be of some different molecules of which
there is no reference in this plate.

CONCLUSION:
The sugar mixture 1 contains glucose, maltose-monohydrate, saccharose, D (-)-Ribose and
Raffinose. Whereas, the sugar mixture 2 contains only glucose and saccharose.
 4. PROTEIN GEL STAINING TECHNIQUES
AIM: To study protein gel staining technique

A] SILVER STAINING
KEY PRINCIPLE:
Silver staining is a very sensitive method for detecting small amounts of proteins and low-
molecular-weight nucleic acids in polyacrylamide gels. The technique involves the deposition of
metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver
nitrate in the staining reagent) interact and bind with certain protein functional groups. The
strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His),
sulfhydryls (Cys), and amines (Lys). Various sensitizer and enhancer reagents are essential for
controlling the specificity and efficiency of silver ion binding to proteins and effective
conversion (development) of the bound silver to metallic silver. The development process is
essentially the same as for photographic film: silver ions are reduced to metallic silver, resulting
in a brown-black color.

REQUIREMENTS:
Fixative 1: 50% methanol + 10% acetic acid +40 ml distilled water
Fixative 2 : Methanol : acetic acid: distilled water (0.5:0.7:98.8)
0.02% sodium thiosulphate : 20mg sodium thiosulphate in 100ml distilled
water 0.2% sodium nitrate: 200mg silver nitrate in 100ml distilled water + 75
μl Formaldehyde (freshly prepared)
2% sodium carbonate: 2% sodium carbonate in chilled water + 50μl formaldehyde + 2ml of
0.02% sodium thiosulphate solution.
2% citric acid solution: 2.1 gm citric acid in 100 ml distilled water
KEY OBSERVATIONS:

RESULTS:
In lane 1 IgG sample treated with β-mercaptoethanol is loaded where we observe two bands of
heavy chain weighing approx. 42 KD and light chain weighing approx. 20 KD corresponding to
the protein marker. In Lane 2 the IgG sample is loaded is not treated with β-mercaptoethanol
shows a single band which is corresponding to the protein marker of approx. 62 KD present in
lane 3. β-mercaptoethanol breaks the disulphide bonds present in the sample.

CONCLUSION:
Silver staining of IgG with β-mercaptoethanol was carried out successfully. By adding the
weights of both the chains we get the approx. weight of the single band in lane 2 which tells that
β-mercaptoethanol has disrupted the IgG molecule in lane 1.
 B] ACTIVITY STAINING: LDH

KEY PRINCIPLE:
Lactate dehydrogenase (LDH) is an oxidoreductase which catalyzes the interconversion of
lactate and pyruvate. It consists of 4 subunits which may be of 2 different types: M (muscle) and
H (heart), also known as A and B respectively. Five different isoenzymes are therefore possible,
depending on the subunit composition. Tissue specific differences in LDH isoenzymes can be
readily detected by the localization of LDH activity in a polyacrylamide gel after electrophoresis
by an activity staining process where the product of the enzymatic reaction is a water insoluble
stain precipitating in the gel where the LDH proteins are located. The staining is based on
coupled enzyme assays carried out on the gel after electrophoresis and is linked to the
disappearance of NADH, which is visualized by fluorescence. The enzyme LDH oxidizes lactate
to pyruvate in the presence of pyridine nucleotide, NAD. The electrons from NADH are
transferred to phenazine methosulphate (PMS), an electron carrier which is added to the staining
mixture. The reduced PMS carries the electrons to the colourless soluble tetrazolium salt,
Nitroblue tetrazolium (NBT) to yield an insoluble blue formazan at the site of enzyme.

REQUIREMENTS:
Sample: Muscle/Liver/Serum sample

Chemicals and Reagents: 30% Acrylamide gel, Tris HCl glycine buffer pH 8.6, TEMED, APS,
1% agar for sealing, Staining solution [Dissolve 5mg Nitroblue tetrazolium salt (NBT) + 0.9
Phenazine methosulphate and 4.8mg NAD in 20ml of D/W. Add 0.7ml Na-lactate and use
immediately for staining]
KEY OBSERVATIONS:

KEY:

1. LDH1- Heart
2. LDH2- Reticular Endothelial System
3. LDH3- Lungs
4. LDH4- Liver

RESULTS:
After slide electrophoresis four bands of LDH was observed. The band at bottom corresponds to
LDH1, the second band is corresponding to LDH2, the third band corresponds to LDH3 and the
fourth and top band corresponds to LDH4. LDH1 is present in the heart, LDH2 is present in
reticular endothelial system, LDH3 is present in lungs and LDH4 is present in liver.

CONCLUSION:
The patient’s serum sample contains all the four isozymes. So this indicates that the patient is
suffering from multiple organ damage.
 5. VISCOSITY STUDIES OF PROTEINS
AIM: To determine the changes in the conformation of BSA/Albumin by
viscosity Measurement
KEY PRINCIPE:

Viscosity, resistance of a fluid (liquid or gas) to a change in shape, or movement of

neighbouring portions relative to one another. Viscosity denotes opposition to flow. The force
required to slip one layer of a fluid past another with a given viscosity is called shearing stress
while the rate of movement is called rate of shear. Resistance to this movement is called the
viscosity. The viscosity of a liquid can therefore be defined as the force per unit area necessary
to maintain a unit velocity between two parallel planes of the liquid separated by a unit
distance. The unit of viscosity is poise. The official S.I. unit is Pascal-second. High
concentration of urea causes denaturation of proteins and loss of conformation by unfolding
due to weakening of bonds that maintain the tertiary structure. This causes the molecule to be
less compact, leading to increase in the viscosity of the solution compared to that of native
protein molecule.

REQUIREMENTS:
Ostwald viscometer

water bath at 300c

potassium chloride-100mM/liter

urea solution (0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 moles/liter in 100mM/liter KCL)

BSA (10g/liter or Egg albumin in 100mmol/liter KCL and the above urea solutions).
KEY OBSERVATIONS:

Sr.No. Conc. of urea t0 t1 t1/to

(M) (sec) (sec) = Relative
viscosity
1 0.5 63.3 65.67 1.04
2 1 68.67 70.67 1.03
3 2 70.67 74.33 1.05
4 3 71 75.33 1.06
5 4 74.67 80 1.07
6 5 77.67 84.67 1.09

SCHEMATIC DIAGRAM:

RESULTS:
Determination of changes in the conformation of BSA/Albumin by viscosity Measurement was
successfully carried out. The graph shows the relative viscosity of a protein against concentration
of urea, the relative viscosity is directly proportional to the concentration.eg. the increase in urea
concentration leads to increase in viscosity, which means the urea denatures the protein or
degrade the protein structure which results in less resistance of protein or increase the fluidity of
the protein which increases the viscosity of protein.

CONCLUSION:
The relative viscosity has been obtained and studied with the help of graph
 6. ISOLATION OF STARCH FROM POTATO AND ITS ESTIMATION BY
ANTHRONE METHOD

AIM: To isolate starch from potato and estimate its quantity by Anthrone method.

KEYPRINCIPLE:
Starch is a polymeric carbohydrate consisting of numerous glucose units joined by glycosidic
bonds called polymers. This polysaccharide is produced by most green plants as energy storage.
The microscopic appearance of starch is in the form of granules. They differ in size depending
on the source from which they were isolated. Starch is insoluble in water and rapidly settles at
the bottom. To estimate the starch quantity anthrone method is used. Anthrone test is a group test
for carbohydrates that provides a rapid and convenient method for quantification of
carbohydrates that are either free or bound to any lipids or proteins. If carbohydrate is present in
the form of free carbohydrate as poly- or monosaccharide or bound as in a glycoprotein or a
glycolipid, the concentrated acid in the Anthrone reagent first hydrolyses it into component
monosaccharide. Similarly, the concentrated acid then catalyzes the dehydration of the
monosaccharides to form furfural (from pentoses) or hydroxyl furfural (from hexoses). furfural
or hydroxyl furfural formed condenses with two molecules of naphthol from the Anthrone
reagent to form a blue-green complex. The complex can then be quantified by measuring the
absorbance of 620 nm wavelength in a spectrophotometer or in a red filter colorimeter .

REQUIREMENTS:
(I) Isolation of Starch
Potato, Muslin Cloth, Watch Glass, Motor and Pestle, Slide, 1% iodine solution

(II) Anthrone Method

Anthrone reagent (0.2% in conc. H2SO4), glucose (0.1g/litre), boiling water
bath, Colorimeter
KEY OBSERVATIONS:

Sr. No. Conc. of glucose Absorbance at

(µg/ml) 620nm
1 20 0.25
2 40 0.29
3 60 0.43
4 80 0.45
5 100 0.49
6 Blank 0.0
7 Unknown 0.40

SCHEMATIC DIAGRAM:
RESULTS:
The amount of starch present in the potato is calculated by the equation: y = mx + c
By determining the graph, substituting the value of y, m and c in the above equation.

y= mx+c
0.40=0.0032x+0.19 (y=0.40)
0.40-0.19=0.0032x
0.21=0.0032x
0.21/0.0032=x
x=65.625
The amount of starch present in the potato was obtained 65.625ug/ml

CONCLUSIONS:
Isolation of starch from potato and its estimation was successfully carried out using anthrone
method and amount of starch present in the potato was obtained 65.625ug/ml
 7. THE ISOLATION AND ASSAY OF GLYCOGEN FROM LIVER AND
SKELETALMUSCLES OF BIRD / MAMMAL

AIM: To isolate glycogen from mammalian liver and estimate its quantity by DNSA method.

KEY PRINCIPLE:

The liver glycogen maintains the level of blood glucose and represents a central reserve of fuel
for the body tissues. In a well-fed animal, glucose is converted to glycogen in the liver by
process of glycogenesis but during starvation the liver glycogen is broken down to glucose by
enzymatic reactions called glycogenolysis and become depleted in about 24 – 48 hrs. Glycogen
is released from the tissue by heating with strong alkali and precipitated by the addition of
ethanol. Sodium sulphate is added as a co-precipitant to give a quantitative yield of glycogen.
The polysaccharide is then hydrolysed in acid and the glucose released estimated. To estimate
the glycogen quantity DNSA method is used. In DNSA method the DNSA reagent that is 3,5-
Dinitrosalicylic acid is used for estimation of reducing sugars. It detects the presence of free
carbonyl group (C=O) of reducing sugars. This involves the oxidation of the ketone functional
group (in fructose) and the aldehyde functional group (in glucose) respectively, by 3,5-
Dinitrosalicylic acid (yellow color) to by 3-amino-5-nitrosalicyclic acid that is ANSA in alkaline
medium.

REQUIREMENTS:
Liver tissue, KOH (30%), saturated Na2SO4, 95% ethanol, Dil. HCl, phenol red indicator,
NaOH (0.5mol/lit), DNSA reagent, centrifuge tubes, boiling waterbath, test tubes, pipettes.
KEY OBSERVATIONS:

Sr. No. Conc. of glucose Absorbance at

(µg/ml) 620nm
1 Blank 0.0
2 400 0.34
3 800 0.44
4 1200 0.63
5 1600 0.81
6 2000 0.99
7 Unknown 0.73

SCHEMATIC DIAGRAM:
RESULTS:
The concentration of the unknown sample is calculated by the equation: y = mx + c
By determining the graph, substituting the value of y, m and c in the above equation.
y = 0.0004x + 0.141
0.73 = 0.0004x + 0.141
0.73 – 0.141 = 0.0004x
0.589 = 0.0004x
x = 0.589/0.0004
x = 1472.5
The unknown concentration “x” was found to be 1472.5µg/ml.
The colour of the reagent changes from yellow to orange or red, depending upon the
concentration of reducing sugar present after incubation.

CONCLUSIONS:
Isolation of glycogen from mammalian liver was successfully carried out. and estimation was
done by using DNSA method. The unknown concentration was found out to be 1472.5µg/ml by
graphical representation.
 8. ANTIMICROBIAL SENSITIVITY TEST AND DEMONSTRATION OF
DRUG RESISTANCE

AIM: To determine the sensitivity of different antimicrobial solutions and demonstrate

drug resistance

KEY PRINCIPLE:
Antibiotic sensitivity testing is a term used to describe the susceptibility of bacteria to
antibiotics. AST is usually carried out to determine which antibiotic will be most successful in
treating a bacterial infection in vivo It is important for detection of MRSA The determination as
to whether the organism is susceptible, intermediate or resistant to an agent is made by
comparing zone sizes obtained The agar diffusion assay is one method for quantifying the
ability of antibiotics to inhibit bacterial growth. Interpretation of results from this assay relies
on model-dependent analysis, which is based on the assumption that antibiotics diffuse freely
in the solid nutrient medium. The agar cup method can also be used for assays of antimicrobial
agents in body fluids like serum, CSF to determine whether therapeutic levels of antimicrobial
agents have reached the likely sites of infection.

REQUIREMENTS:

Sterile Mueller Hilton agar butt (20ml)

Sterile Petriplates – l
Sterile 1ml pipette - 4
Standard antibiotic
solutions
Sterile Cork borer (external diameter l0 mm)
 KEY:
KEY OBSERVATIONS: A= std. inhibitory drug

1. Gentamycin

2. Chloramphenicol

3. Ampicillin

4. Tetracycline

5. Oxacillin

6. Amoxycillin

Test Organism: S.aureus

RESULTS:
Antimicrobial sensitivity test and demonstration of drug resistance was successfully carried out
using agar cup diffusion assay. In ditch 1, 2, 4 zone of inhibition was observed that indicates the
drug gentamicin, chloramphenicol, Tetracycline are sensitive against s.auerus. in ditch 3, 6,
5 zone of inhibition does not observed indicates that the drug ampicillin, oxaicillin, amoxyicillin
are resistance against s.aureus. the drugs which are resistance against s.aureus this drugs belong
to the cillin family. Although the mechanisms of the drugs vary, generally they fight infections
by attacking the walls of bacterial cells.

CONCLUSIONS:
The antibiotic sensitivity test using diffusion assay was performed against organism was most
sensitive to gentamycin, chloramphenicol, tetracycline this means antibiotics is most effective
against the bacteria may be an appropriate choice for treatment. While the organisms is resistant
to ampicillin, oxacillin, amoxicillin which means this is a sign of ineffective drug may not be a
appropriate choice for treat
 9. CHROMOSOMAL ABNORMALITIES

Spectral Karyotyping (SKY):

Spectral karyotyping is a molecular cytogenetic technique that allows differential visualization of

all human chromosomes in distinct colours with a single hybridization and image exposure.
Spectral karyotyping (SKY) involves the use of 24-color, whole chromosome- painting
permitting visualization of each chromosome in one experiment. This technology is based on the
principles of spectral imaging and Fourier spectroscopy. Image acquisition is accomplished by
conventional fluorescence microscopy and the use of a specially designed triple filter (SKY
CUBETM, Applied Spectral Imaging). The method of spectral karyotyping (SKY) is based on a
combination of the technologies of charge-coupled device imaging and spectrometry. The
engineering feasibility has been realized in the SpectraCube system from Applied Spectral
Imaging Inc., and it allows the simultaneous identification of all 24 human chromosomes.
Applications for SKY include pre- and postnatal characterization of certain numerical and
structural rearrangements and complex karyotypes and highly informative analysis of sample
materials with only single or few cells available for investigation.

Comparative Genomic Hybridization (CGH):

Comparative genomic hybridization (CGH) is a technique that permits the detection of
chromosomal copy number changes without the need for cell culturing. An advanced method to
analyze chromosome and gene modifications is comparative genomic hybridization (CGH),
which can be used for genome-wide analysis. In its original form, DNA is isolated and
fragmented from an experimental sample and a control sample. Both samples are then labelled
with two different fluorescent colours, usually green and red. The DNA samples are mixed and
the generated probes compete for hybridization on a normal chromosome. If both samples are
identical, a mixed yellow/orange colour is seen everywhere. A red signal indicates an
overrepresentation of the red-labelled experimental DNA, the result of gene duplication. A green
signal, on the other hand, indicates a gene loss. CGH allows the identification of gene or
sequence deletions and amplifications in a very precise manner. In one single experiment, many
thousand sequences are analyzed simultaneously. CGH compares the patient’s genome against a
reference genome and identifies differences between the two genomes and hence locates regions
of genomic imbalance (copy number variations (CNVs) in the patient.
 1. TO PREPARE A HEAT KILLED T.A.B VACCINE.

AIM: To prepare heat killed T.A.B. Vaccine

KEY PRINCIPLE:
A vaccine is biological preparation that provides active acquired immunity to a
particular disease. A vaccine typically contains an agent that resembles a disease-causing
microorganism and is often made from weakened or killed forms of the microbe, its toxins,
or one of its surface proteins. A heat killed vaccine of the bacteria is one whereby the
virulence of bacteria is destroyed by heat treatment but the antigenicity of the strain is
preserved. This is achieved by heating bacterial suspension at relatively low temperature for
longer period e.g. 60° C for 1 hour.
In case of T. A. B. vaccine they are mixed in following proportion:
1 x 109 cells/ml of S. typhosa
7.5 x 108 cells/ml of S. paratyphi A

7.5 x 108 cells/ml of S. paratyphi B

The vaccine prepared should be free of any viable organisms or chemical contaminants which
may give rise to an allergic reaction in the body. Hence, strict sterility control measures should
be maintained, while preparing vaccines.

REQUIREMENT
Sterile nutrient agar slants, sterile saline, Sterile 1 ml & 10 ml pipettes, Water bath, thermometer,
24 hr old culture of bacteria (S.typhosa, S.paratyphi A, S.paratyphi B), Set of Brown‘s opacity
tubes.

KEY OBSERVATIONS:
Inoculated nutrient agar slants with bacterial cultures S.typhosa, S.paratyphi A, S.paratyphi B
was obtained in pure cultural growth and the cultures were further used to determine the TDP
and TDT for all the three cultures.
FLOWCHART:

S.typhosa S.paratyphi A S.paratyphi B

Inoculate nutrient agar slants with bacterial

cultures, incubate at 37°C for 24hrs

Wash the culture of the slant using

sterile saline, adjust the density with the
help of Brown‘s opacity tubes.

Determine the TDP and TDT of all

the three cultures. Heat the culture at
their respective TDP and TDT.

Mix equal volume of all the three

cultures. Use this sample as heat killed
vaccine in check its sterility.

RESULTS:
The preparation of TAB vaccine was carried out successfully. The results indicates pure culture
formation of S.typhosa, S.paratyphi A, S.paratyphi B and further the Thermal Death Point (TDP)
and Thermal Death Time (TDT) were calculated.

CONCLUSION:
The preparation of TAB vaccine was carried out successfully with fixed proportion of S.typhosa,
S.paratyphi A and S.paratyphi B. The Thermal Death Point (TDP) and Thermal Death Time
(TDT) were determined to be 51oC for 10 minutes, 61oC for 4 minutes and 61 oC for 4 minutes,
for S. typhosa, S. paratyphi A and S.paratyphi B respectively. On performing the sterility test for
vaccine, it resulted with no growth seen in the test i.e., absence of turbidity. This states that the
bacterial vaccine is in sterile form and can be further used.
 DETERMINATION OF TDP AND TDT

AIM: To determine the thermal death point and thermal death time of S. typhosa, S. paratyphi A,
S.paratyphi B

KEY PRINCIPLE:
The heat resistance of micro-organisms usually is expressed in terms of their Thermal death time
(TDP) and Thermal death time (TDT). The thermal death point (TDP) of a microorganism is the
lowest temperature at which all microbes are killed in a 10-minute exposure. The thermal death
time (TDT), is the length of time needed to kill all microorganisms in a sample at a given
temperature.

REQUIREMENTS:
Sterile suspension tubes, Sterile l ml pipettes, Sterile recovery tubes containing sterile
nutrient broth, Sterile saline, Thermometers, Water bath.

KEY OBSERVATIONS:

Microbes TDP TDT

S. typhosa 56oC 10 minutes
S.paratyphi A 61oC 4 minutes
S.paratyphi B 61oC 4 minutes

FLOWCHART:

A] Determination of TDP B] Determination of TDT

Pipette 0.5ml aliquots of the Pipette 0.5ml aliquots of the
saline suspension of each culture saline suspension of each culture
into 7 sterile suspension tubes, into 7 sterile suspension tubes,
each for incubation at variation each for different duration of
temperature range incubation

Adjust the temperature of the

Adjust the temperature of
water bath in an ascending order
the water bath to a
70°C, 75°C, 80°C, 85°C, 90°C,
temperature determined in
and 95°C and immerse 1 tube at
the previous experiment i.e.
a time for 10 min. at each
TDP
temperature
 Immerse all the suspension
Cool the tubes and transfer the
tubes and remove one tube at
contents to respective recovery
regular intervals of time, e.g.3.
tubes which contain 4.5ml of
6, 9,12,15,18 minutes.
Sterile Nutrient Broth as a
recovery reagent

Cool the tubes and

Incubate the tubes at 37°C for 24 transfer the contents to
hrs. respective recovery tubes
which contain 4.5ml sterile
Tabulate the results and Nutrient Broth as a
determine the thermal death recovery reagent.
point for the culture.
Incubate the tubes at 37°C for 24
hrs and check the growth. The
tube showing absence of growth
of the culture would be the tube
used for determining TDT
RESULTS:
The values of Thermal Death Point (TDP) and Thermal Death Time (TDT) of microbes was
found to be 1oC for 10 minutes, 61oC for 4 minutes and 61oC for 4 minutes, for S. typhosa, S.
paratyphi A, S.paratyphi B respectively.

CONCLUSIONS:
Determination of Thermal Death Point (TDP) and Thermal Death Time (TDT) of S. typhosa, S.
paratyphi A, S.paratyphi B was successfully carried out. The Thermal Death Point (TDP) and
Thermal Death Time (TDT) were determined to be 51oC for 10 minutes, 61oC for 4 minutes and
61oC for 4 minutes, for S. typhosa, S. paratyphi A, S.paratyphi B respectively. These
temperatures and period will inform us the temperature amount at which the spoilage causing
organisms are destroyed and time taken to kill the micro-organism.
 TO CHECK THE STERILITY OF THE BACTERIAL VACCINE

AIM: To check the sterility of the bacterial vaccine.

KEY PRINCIPLE:
Sterility can be defined as the freedom from the presence of viable microorganisms. However,
the conditions that guarantee absolute sterility are usually too harsh for active ingredients, and
the definition of sterility for a medicinal product must be defined in functional terms. sterility
testing is required to ensure viable contaminating microorganisms are not evident in a product.
This testing is conducted by direct inoculation or membrane filtration methods and can be
performed in an isolator or cleanroom environment.
The media commonly used for sterility testing are:

Nutrient Broth: Suitable for the aerobic microorganisms. Oxidation-reduction potential value of
this medium happens to be quite high to enable the growth of the anaerobes specifically.

Cooked Meat Broth or Thioglycollate Broth: Used for Clostridia and other anaerobic bacteria. It
essentially comprises of the following ingredients, namely Glucose and Sodium thioglycollate-
that invariably serve as: an inactivator of mercury compounds, to augment and promote reducing
parameters, and an oxidation-reduction indicator and agar to cause reduction of the ensuing
convection currents.

Sabourauds Broth: It is used for fungal species. It essentially bears two vital and important
characteristic features, such as, an acidic medium and contains a rapidly fermentable
carbohydrate e.g., glucose or maltose.

Nutrient Broth+ Paraffin oil overlay: It is used as a medium for anaerobic bacterial growth, as
the paraffin oil overlay disconnects the interaction of medium with environment.

Sterility tests are exclusively based upon the principle that in case the bacteria are strategically
placed in a specific medium that caters for the requisite nutritive material and water, and
maintained duly at a favourable temperature (37 ± 2°C), the microbes have a tendency to grow,
and their legitimate presence may be clearly indicated by the appearance of a turbidity in the
originally clear medium.
REQUIREMENTS: Sterile nutrient broth tubes, sterile sodium thioglycollate medium broth
tubes, sterile Sabouraud‘s broth tubes, Vaccine sample, 24 hr old cultures of S. aureus, C.
albicans, E.coli, B.subtilis, C.perfringens as standard cultures.

OBSERVATION:

Test No. Tests conducted Observation

1 Positive Control (PC) Growth

2 Negative Control (NC) No growth

3 Inhibition Control (IC) Growth

4 Test No growth

5 Refrigerator Control (RC) No growth

FLOWCHART:

KEY: PC - Positive Control, NC - Negative Control, IC - Inhibition Control, RC- Refrigeration Control

Perform for all the three cultures of salmonella

Incubate all the tubes except refrigeration control at 37°C

for 24hrs, Incubate RC tube in refrigerator for 24 hrs
No. MEDIUM PC NC IC TEST RC
1 Nutrient broth Medium+1 Medium Medium+1 Medium Medium +
loopful loopful +0.1 ml 0.1 ml
std.culture i.e. std.culture+0.1 vaccine vaccine
B.subtilis ml vaccine (Refrigerator)
2 Thioglycolate Medium+1 Medium Medium+1 Medium Medium +
broth loopful loopful +0.1 ml 0.1 ml
std.culture i.e. std.culture+0.1 vaccine vaccine
C.perfringens ml vaccine (Refrigerator)
3 Sabouraud‘s broth Medium+1 Medium Medium + Medium Medium +
loopful 1 loopful std. +0.1 ml 0.1ml
std.culture i.e. culture + vaccine vaccine
C.albicans 0.1 ml vaccine (Refrigerator)

RESULTS:
There are five tests been carried out, consisting of a positive control,
negative control, inhibition control, refrigerator control and a test. In the
positive control and inhibition control growth was observed. In the
negative control, test, refrigerator control there is no growth.

CONCLUSION:

The sterility checking of bacterial vaccine was successfully carried out. the result observed is no
growth seen in the test i.e., absence of turbidity. This states that the bacterial vaccine test sample
is in sterile form and can be further used.
 2. SERUM ELECTROPHORESIS

AIM: To perform fractionation of Serum Proteins by Horizontal Electrophoresis

KEY PRINCIPLE:
The serum protein electrophoresis (SPEP) test measures specific proteins in the blood to help
identify some diseases. Proteins are substances made up of smaller building blocks called amino
acids. Proteins carry a positive or a negative electrical charge, and they move in fluid when
placed in an electrical field. Serum protein electrophoresis uses an electrical field to separate the
proteins in the blood serum into groups of similar size, shape, and charge. Blood serum contains
two major protein groups: albumin and globulin. Both albumin and globulin carry substances
through the bloodstream. Using protein electrophoresis, these two groups can be separated into
five smaller groups (fractions):
Albumin: Albumin proteins keep the blood from leaking out of blood vessels. Albumin also
helps carry some medicines and other substances through the blood and is important for tissue
growth and healing. More than half of the protein in blood serum is albumin.
Alpha-1 globulin: High-density lipoprotein (HDL), the "good" type of cholesterol, is included in
this fraction.
Alpha-2 globulin: A protein called haptoglobin, which binds with hemoglobin, is included in the
alpha-2 globulin fraction.
Beta globulin: Beta globulin proteins help carry substances, such as iron, through the
bloodstream and help fight infection.
Gamma globulin: These proteins are also called antibodies. They help prevent and fight
infection. Gamma globulins bind to foreign substances, such as bacteria or viruses, causing them
to be destroyed by the immune system.
Each of these five protein groups moves at a different rate in an electrical field and together form
a specific pattern. This pattern helps identify some diseases.
REQUIREMENTS:
Chemical & reagents: 1%Agarose gel

Tris glycine buffer (ph8.4) [tris-6g, glycine-28.8g, D/W-1000ml]

Loading dye:10μg of 0.01% bromophenol blue + 30μg of 40% glycerol

Staining solution (0.125% CBB + 50% methanol + 10% acetic acid)

Destaining solution (50% methanol + 10% acetic acid)

Glasswares: Clean grease free slides (2), Cover slips (1), 10 ml pipette (1), Conicalflask ((10)

Instruments: Horizontal electrophoresis unit with power pack, Glass slides and glass capillaries

Miscellaneous: Whatman filter paper No 1strips (2), Filter paper wicks, Petri plates, Plastic box

(1), Hot air oven (1)

KEY OBSERVATIONS:

FLOWCHART:

Weigh 0.4g of agarose and place it in a conical flask.

Add about 50ml of Tris buffer and place in a boiling water bath for dissolving

Layer about 2.5ml of agarose on each three glass slides

After allowing the gel to set, apply the serum with the help of cover
slip about 1-1.5cm away from the edge of the slide
 Add the Tris buffer in the anode and cathode compartments till about
3/4th its capacity and place the slides in the tank

Connect the slides to the buffer by the Whatman‘s No1 filter paper wicks

Connect to power supply and set the voltage to 200v.After 3hrs put off the power
supply

The run can be stopped earlier depending upon the movement of the tracker dye

Keep the slides for staining in staining solution for 1hr

Destain by using destaining solution till the background of the slides becomes clear

Dry the slides at room temperature.

RESULTS:

Sr. No. Fractions % Concentration Ref. rang

1 Albumin 29.5 2.09- 3.20-5.30 (g/dL)
2 Alpha 1 (α1) 7.0 0.50+ 0.10-0.40 (g/dL)
3 Alpha 2 (α2) 10.9 0.77 0.40-1.00 (g/dL)
4 Beta (β) 14.9 1.06 0.50-1.10 (g/dL)
5 Gamma (γ) 37.7 2.68+ 0.70-1.70 (g/dL)
CONCLUSION:
Fractionation of serum proteins by horizontal electrophoresis was carried out successfully. The
result indicates that the % of albumin was found to be 29.5% which is less than a reference range. A
decreased level of albumin is common in many diseases and is especially important in liver
disease. The % of Alpha 1 (α1) was found to be 7.0% which is more than the reference value.
This increase appears in severe alcoholics and in women during pregnancy. The % Alpha 2 (α2)
and Beta (β) was found to be 10.9% and 14.9% respectively which is within the reference range.
The % of Gamma (γ) was found to be 37.7% which is more than its reference value.. The
common causes of hypergammaglobulinaemia (elevated gamma proteins) are severe infections,
chronic liver disease, systemic lupus erythematosus etc. Absence of gamma globulin is called as
agammaglobulinaemia.
 3. LATEX BEAD AGGLUTINATION/PRECEPITATION TEST FOR DETECTION
OF RHEUMATOID FACTOR (RF)

AIM: latex bead agglutination/precepitation test for detection of rheumatoid factor (RF)

KEY PRINCIPLE:
Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints and
other areas of the body. The “rheumatoid factor” is a factor is an antibody that can be found in
the blood of 80% of people with rheumatoid arthritis. In Latex bead agglutination method mixes
blood with tiny rubber latex beads that are covered with human antibodies. If RF is present, the
latex beads clump together agglutinate. This method is best used as a first-time screening test for
rheumatoid arthritis.

REQUIREMETNS:

Latex Reagent, Positive Control Serum, Test slides, Mixing sticks, Plastic Droppers, Glass
Dropper, Fresh Serum.

KEY OBSERVATIONS:

There are two tests has been carried out, Consisting of positive test and negative test. After
performing the following protocol, it will show the presence of agglutination and absence of
agglutination.

FLOW CHART:

25µl of sample or control+ 25µl of RF latex Reagent

 Mix latex reagent well

Rock the slide

Place the slide on an automated rotator at 100 rpm

Observe for agglutination up to 2 minutes

RESULTS:

In positive test (A) agglutination was observed that indicates presence of rheumatoid factors in
significant quantities, whereas in negative test (B) there is no agglutination that indicates absence
of rheumatoid factor.

CONCLUSION:

Latex bead agglutination test for detection of rheumatoid factor was carried out successfully.
Agglutination of red blood cells in the given result indicates positive identification of the blood
antigens whereas, negative test shows the patient does not have the antibody in their serum. This
method is best used as a first-time screening test for rheumatoid arthritis.
 4. SEPARATION OF LYMPHOCYTES ON FICOL HISTOPAQUE AND
VIABILITY COUNT

AIM: To perform density gradient centrifugation for the separation of lymphocyte from
peripheral blood using Ficoll hypaque

KEYPRINCIPLE:

Ficoll density gradient centrifugation is one of the most commonly used method for isolation and
enrichment of human mononuclear cell, particularly from peripheral blood and other biological
fluids. Human lymphocyte can be isolated most readily from peripheral blood. A pure population
of lymphocytes can be obtained by ficoll and ficoll hypaque. From this method after
centrifugation we separate four different layer the polymorphonuclear cells and RBC settle down
at bottom and above this ficoll, PBMC, plasma layer get. Then isolated mononuclear layer
stained by tryphan blue it turns dead cells blue and viable cells unstained.

REQUIREMENTS:
Anticoagulated blood

Sigma Histopaque (Ficoll Hypaque)

Phosphate buffered saline (PBS) pH7.4

Sliconised centrifuge tubes

Pasteur pipettes

KEY OBSERVATION:

Tryphan blue stained cells Viabiity count

 Addition of 2.5ml of Ficoll Before After centrifugation
hypaque centrifugation 15000rpm for 30min

Diluted
peripheral
Diluted blood
peripheral
blood

Remove the lymphocytes ring at interface with the help of Pasteur pipettes, dilute the
mononuclear cells and centrifuge it ,wash with PBS and again centrifuge it and add
tryphan blue followed by application of sample on hemocytometer
RESULTS:
%Cell Viability = [Total Viable cells (Unstained) / Total cells (Viable +Dead)] X 100.

= 657/878 X 100

=74.829%

The %Cell Viability was found to be 74.829%.

CONCLUSIONS:
As %Cell Viability was found to be 74.829% the isolated lymphocytes can further be used for
other experimental analysis.
 5. IN-VITRO DEMONSTRATION OF PHAGOCYTOSIS AND
CALCULATING PHAGOCYTIC INDEX

AIM: To demonstrate the in-vitro phagocytosis and calculate phagocytic index

KEY PRINCIPLE:

Phagocytosis, process by which certain living cells called phagocytes ingest or engulf other cells
or particles. The phagocyte may be a free-living one-celled organism, such as an amoeba, or one
of the body cells, such as a white blood cell. In some forms of animal life, such as amoebas and
sponges, phagocytosis is a means of feeding. In higher animals phagocytosis is chiefly a
defensive reaction against infection and invasion of the body by foreign substances (antigens).
Phagocytosis index is a measure of phagocytic activity determined by counting the number of
bacteria ingested per phagocyte during a limited period of incubation of a suspension of bacteria
and phagocytes in serum. Phygocytic index (n) is the average number of bacteria ingested per
leukocyte in an incubated mixture of normal or immune serum, bacteria, and normal leukocytes.
Phygocytic index = No. of macrophages containing at least one bacterium ÷Total no. of
macrophages counted ×Mean no. of bacteria per positive cell

REQUIRMENTS:
(1) Sample-Human blood ( mononuclear cells, granulocytes-after treatment with Ficoll-Hypaque)

(2) Culture supension

(3) Reagents- RBC lysis buffer (0.037g of EDTA, 0.084g of sodium bicarbonate, 0.82g of
ammonium chloride, mix in 100ml D/W, Phosphate buffered saline (pH 7.4), Field’s A and
Field’s B stains, Methanol

(4) Glassware- Cavity slide, Coverslip

(5) Miscellaneous- Microscope with 100X, Centrifuge machine, Centrifuge tubes, Microfuge
tubes, Vaseline, cedar wood oil
 KEY OBERVATIONS:

SR NO MACROPHAGE NUMBER OF
NUMBER ENGULFED
CELLS
1 1 5
2 2 8
3 3 2
4 4 11
5 5 6
6 6 1
7 7 0
8 8 0

FLOW CHART:

Collect the layer of mononuclear cells and granulocytes obtained after separation using Ficoll
Histopaque in 2 different tubes

A] Mononuclear cells: B] Granulocyte

Take 3 microfuge tubes

Yeast E. coli S.aureus

Tube containing granulocyte

Add RBC lysis buffer

Centrifuge and discard the supernatant

Suspend the pellet in 100μl phosphate

In each tube add 50μl of mononuclear cells and buffered saline
10 of respective cultures

Incubate all the tubes at 37C for 30-45 min

Add 50μl each in 2 microfuge tubes, add
10μl of yeast in each tube
 For viability testing place a drop of For viability testing place a drop of
incubated culture suspension on a coverslip, incubated culture suspension on a coverslip,
place the cavity slide on the coverslip and place the cavity slide on the coverslip and
observe under 45X observe under 45X

Prepare smear of the respective culture, Prepare smear of the respective culture, stain
stain the slide with Field’s B and then the slide with Field’s B and then Field’s A,
Field’s A, wash then dry it and observe wash then dry it and observe
under oil immersion lens

RESULTS:
Phagocytic index = No. of macrophages containing at least one bacterium ÷Total no. of

macrophages counted ×Mean no. of bacteria per positive cell

= 6÷8×33÷6

= 4.12

The phagocytic index was found to be 4.12

CONCLUSION:
In vitro demonstration of phagocytosis was carried out successfully and the phagocytic index
was calculated. The result indicates the phagocytic cells engulfed the bacteria which showed
positive activity and phagocytic index was calculated, it was found to be 4.12 which concludes
that each phagocyte has ingested 4.12 bacteria on an average.
 6. CELL PERMEABILITY TESTING- OSMOTIC FRAGILITY

AIM: Determination of osmotic fragility of red blood cells

KEY PRINNCIPLE:

The osmotic fragility test (OFT) is used to measure erythrocyte resistance to hemolysis while
being exposed to varying levels of dilution of a saline solution. When erythrocytes are exposed
to a hypotonic environment, water enters the cell and causes swelling and eventual lysis. The
susceptibility of osmotic lysis of erythrocytes is a function of surface area to volume ratio. The
osmotic fragility test is used to gauge the level of hemolysis in a collected sample of a patient’s
blood, and this is compared to a control sample. An osmotic fragility test can be used to help
diagnose two hereditary conditions thalassemia and hereditary spherocytosis.

REQUIRMENTS:

Test tubes-15
10ml and 1 ml pipette
Colorimeter with a filter for 540mn
1 g/dl buffered sodium chloride solution
Distilled water
5% cell suspension
KEY OBSERVATION:

Tube no. Conc. Of Absorbance at Hemolysis%

working 540nm
solution
1 0.1 0.846 99.41
2 0.2 0.823 96.70
3 0.3 0.745 87.54
4 0.35 0.684 80.37
5 0.4 0.561 65.92
6 0.45 0.425 49.94
7 0.5 0.247 29.02
8 0.55 0.157 17.62
9 0.6 0.067 7.87
10 0.7 0.032 3.76
11 0.8 0.018 2.11
12 0.0 0.851 100
KEY:

Normal person- close to 0.45% or slightly lower

Hereditary Sphaerocytosis - 0.5%

Sickle Anaemia - 0.36%

Thalassaemia - 0.35 to 0.4%

As a control, parallel run- should be done with a normal blood speciemen

FLOW CHART:

Take 2 ml of blood in a graduated centrifuge tube and centrifuge at 1500 rpm for 10min

Discard the supernatant, add 2-3 ml of l g/dl saline mix and centrifuge at 1500
rpm for 10min

Repeat the 2nd step 3 times. Discard the supernatant, measure the volume of the packed
cells

Dissolve the pellet in equal quantity of l g/dl saline, make upto 20ml.this gives rise to
5% cell suspension

Add l ml of 5% cell suspension to each of the 12 working solutions of buffered saline

Mix well and keep all the tubes at room temperature for 30min, Mix again and
centrifuge at 1500rpm for l0 min

Read absorbance of the supernatant of each tube at 540nm by setting blank to 100%T

Calculate the percent Haemolysis and Draw the fragility curve

Determine the median corpuscular fragility (MCF) which causes 50% lysis
RESULTS:
Determination of osmotic fragility of red blood cells was successfully carried out. Osmotic
fragility was performed for determining percentage haemolysis (% haemolysis) upon increasing
the concentration of saline for achieving Median corpuscular fragility (MCF) 0.45% of saline
was used.

CONCLUSION:
Osmotic fragility was performed for determining percent Haemolysis (% Haemolysis). The MCF
was determined to be 0.45%. Comparing the results or obtained value with the Standard values,
it can be concluded that the individual is not suffering from any disorder/disease.
 7. ISOLATION OF CELL ORGANELLE BY DIFFERENTIAL
CENTRIFUGATION TECHNIQUES FROM PLANT / ANIMAL
SOURCES

AIM: To isolate cell organelle by differential centrifugation techniques

KEY PRINCIPLE:
Eukaryotic cells contain several types of intracellular membrane-bound structures known as
organelles which perform a variety of specific functions. For example, mitochondria synthesize
ATP; chloroplasts convert light energy to chemical energy, while the endoplasmic reticulum is
involved in protein synthesis. An ubiquitous first step in the isolation of subcellular organelles is
to homogenize the cells. This process involves breaking open the cell membrane. Cell
homogenization can be achieved through various methods including dounce homogenization,
filtration, grinding, sonication, solubilization. The method of choice depends on the type of
tissue to be homogenized and the specific purpose of the experiment. In homogenization process
isotonic sucrose is added to the homogenization buffer to prevent osmotic rupture of organelle
membranes since it is usually important to purify intact organelles. Once the cellular extract is
formulated by homogenization differential centrifugation is applied. Differential centrifugation
works by a stepwise increase in the centrifugation speed. Lower speeds at the beginning are us ed
to eliminate the heavier particles from the sample, and the speed is then increased until the targets
themselves are pelleted. This sedimentation results from the interaction between a particle's weight and
the resistance it encounters in moving through a suspension medium and the relative centrifugal force
exerted on the particle. particles that are relatively large or dense will sediment more rapidly than
particles that are smaller and lighter. With respect to the major components found in cells, the order of
sedimentation is typically from most to least dense: nuclei, mitochondria, lysosomes, plasma membrane,
endoplasmic reticulum, and contractile vacuoles. Depending on the specific cell type, however, this order
can vary. Additionally, differences in the rate of sedimentation are sometimes not large enough to provide
separation of one organelle from another.

REQUIREMENTS:
polycarbonate nucleopore filters, microfuge tubes, isotonic sucrose, centrifuge, animal/plant
tissue sample.
KEY OBSERVATIONS:

Enzymes organelle Reactants Positive Absorbance

Succinate Mitochondria Succinate NBT Purple 630nm
dehydrogease
Acid phosphatase Lysosomes p-nitrophenol Yellow 410nm
phosphatase
Alkaline Contratile p-nitrophenol yellow 410nm
phosphodiesterase vacuole thymidine 5’
monophosphatase

SCHEMATIC DIAGRAM:
RESULTS:
Isolation of cell organelles was successfully carried by differential centrifugation from plant /
animal source. For succinate dehydrogenase assay purple colored was observed at 630nm. Acid
phosphatase assay yellow colored was observed at 410nm. Alkaline phosphodiesterase yellow
colored was observed at 410nm.

CONCLUSION:

As result indicates for succinate dehydrogenase assay purple colored was observed at 630nm.
Acid phosphatase assay yellow colored was observed at 410nm. Alkaline phosphodiesterase
yellow colored was observed at 410nm we can concluded that the cell organelles was isolated
successfully by using differential centrifugation method.
 8. ISOLATION OF MITOCHONDRIAL DNA

AIM: To isolate mitochondrial DNA

KEY PRINCIPLE:
Most eukaryotic cells contain hundreds of mitochondria, with hundreds of copies of mtDNA
(estimated 2-10,000 mtDNA copies per cell). Because of this, it is common for individual
mutations to be present in only a subset of mitochondria. Mitochondria produce the majority of
adenosine triphosphate (ATP) that is required to power a multitude of cellular processes. These
cellular powerplants have a 16-kb circular genome that encodes a handful of proteins, and
mutations in mitochondrial DNA (mtDNA) are associated with a wide range of diseases,
including hearing loss, muscle disorders, diabetes, neurodegenerative disease, and cancer. Before
you can sequence mtDNA to study the effect of these mutations in these and other disorders, you
need first to isolate it. mtDNA differs from nuclear DNA in several essential ways. There are
various methods available for enriching mtDNA, but before choosing one, you should consider
the, Purity and enrichment levels you require for your downstream analysis, The time you have
available to spend on the enrichment process, Level of skill and mastery required to perform the
method

REQUIREMENTS:
5X Cytosol buffer
Mitochondria lysis
buffer Enzyme mix
TE buffer
KEY OBSERVATIONS:

\
Fig 1.Isolation of mitochondria

FLOW CHART:

A] Isolation of mitochondria B] Isolation of mitochondrial DNA

Collect cells by Lyse the mitochondria

centrifugation at 600 x g in Mitochondrial Lysis
for 5 minutes at 4°C. Buffer, keep on ice for
10 minutes.

Wash cells with 5-10

ml of ice-cold PBS, Add Enzyme Mix and
Centrifuge for 5 min at incubate at 50°C water
4°C. Remove bath for 60 minor
supernatant. longer until the
solution becomes clear.
Re-suspend cells in Cytosol Add absolute ethanol, mix
Extraction Buffer, Incubate and keep at -20°C for10
on ice for 10 minutes minutes.

Homogenize cells in an ice- Centrifuge in

cold dounce tissue grinder. microcentrifuge at top
speed for 5 min at room
Transfer homogenate to temperature.
a 1.5 ml
microcentrifuge tube, Remove the supernatant,
centrifuge for the pellet is mitochondrial
10minutes at 4°C. The DNA
step removes nuclei
intact cells
 Wash the DNA pellet with
Transfer supernatant to a ethanol, Remove the trace
fresh tube, centrifuge for 30 amount ethanol, Air dry
mins at 4°C, Remove for 5 min
supernatant

Re-suspend the DNA

Re-suspend the pellet in 1 in buffer or water,
ml Cytosol Extraction Store the extracted
Buffer, centrifuge at for 30 DNA at -20°C for
mins at 4°C future use

Remove the supernatant,

the pellet is isolated
mitochondria

RESULTS:
First the isolation of mitochondria was carried out. In mitochondria isolation method after last
centrifugation pellet was observed in tube which is mitochondria. As shown in fig1.the tube
contain other contaminants and intact mitochondria. the intact mitochondria are taken for further
studies such as mitochondrial DNA isolation. In mitochondrial DNA isolation the DNA band
visualize on a agarose gel. In the fig2. band was observed that indicate the isolation of
mitochondrial DNA was successfully carried out.

CONCLUSION:
In this study the isolation of mitochondrial DNA was successfully carried out by gradient
centrifugation and visualizing DNA band on agarose gel. Isolation of mitochondrial DNA can
provide wealth information of wide range of diseases, including hearing loss, muscle disorders,
diabetes, neurodegenerative disease, and cancer.
 9. ISOLATION OF CHLOROPLAST DNA

AIM: To isolate chloroplast DNA

KEY PRINCIPLE:
Chloroplasts serve as important cytoplasmic organelles in higher plants. Multiple
biochemical processes take place in chloroplasts, including photosynthesis, nitrogen
metabolism, sulfate reduction, and synthesis of starch, amino acid and lipid. As semi-
autonomous cellular organelles, chloroplasts possess their own independent genome
and a full complement of transcription and translation machinery to express their
genetic information Two experimental methods are often employed to collect cpDNAs
in plants. The first is the whole chloroplast genome amplification from total DNA
using long polymerase chain reaction (PCR), and the second is direct isolation of
cpDNAs from fresh plant materials based on sucrose gradient. cpDNA isolation from
fresh plant materials uses high-salt buffers, a sucrose density gradient or Percoll
gradient to separate chloroplast first, and then DNase to remove nuclear DNA.

REQUIREMENTS:
Cold isolation buffer: 1.25 M NaCl, 50mM Tris-HCl (pH 8.0), 5mM EDTA, 0.1%
BSA (w/v), 0.1%b-mercaptoethanol (v/v).
KEY OBSERVATIONS:

Cell debris

Intact
chloroplast

KEY OBSERVATIONS
Fig 1.Isolation of mitochondria
Fig 2.Chloroplast DNA visualization in
agarose gel
FLOW CHART:

A] Chloroplast isolation: B] Chloroplast DNA isolation:

20g leaves
collected, kept in
Add 1/10 volume of
dark 48 -72hr at
10% CTAB, Incubate
4uC
at 55uC for 1 to 2

Extract the supernatant

Cut leaves into with saturated phenol and
pieces chloroform:isoamyl-
alcohol (24:1) in the
centrifugation of 10000 g
20 min for twice.

Blend/homogenized Add isopropyl alcohol to

in buffer for 30S the upper clear aq. phase
put centrifuge bottles in
the -20OC for 1 hr or
overnight

Centrifuge the aq.Phase,

Filter homogenate
using miracloth
Centrifuge
homogenate 3000g
for 10mins
Resuspend the pellet
in 30ml of buffer, and
repellet the chloroplast
cpDNA pellet is washed
with ethanol, air-dried,
and re-dissolved in
50ml TE buffer
Treat the cpDNA
sample with 2ml
RNAse, visualize
DNA band on a
0.8% agarose gel.
 Resuspend the final
pellet in buffer

RESULTS:
First the isolation of chloroplast was carried out using sucrose gradient method. In chloroplast
isolation method after last centrifugation green color pellet was observed in tube. As shown in
fig1.the tube contain cell debris and intact chloroplast. the intact chloroplasts are taken for
further studies such as chloroplast DNA isolation. In chloroplast DNA isolation the DNA band
visualize on a agarose gel. In the fig2. band was observed that indicate the isolation of
chloroplast DNA was successfully carried out.

CONCLUSION:
In this study the isolation of chloroplast DNA was successfully carried out by using sucrose
gradient centrifugation and visualizing DNA band on agarose gel. Isolation of chloroplast DNA
can provide wealth information for researchers on plant phylogeny, molecular ecology,
comaparitive genomics, population genetic and evolution and also contribute to chloroplast
transformation for crop improvement.
 10. CELL DEATH /APOPTOSIS STUDIES USING FLOW-CYTOMETRY

KEY PRINCIPLE:
Apoptosis is the process of programmed cell death. Apoptosis removes cells during
development. It also eliminates pre-cancerous and virus-infected cells, although “successful”
cancer cells manage to escape apoptosis so they can continue dividing. Apoptosis maintains the
balance of cells in the human body and is particularly important in the immune system. Flow -
cytometry is one of the most popular and versatile applications for studying apoptosis. It offers
the ability to study large numbers cells individually rather than a mixed population, examining
simultaneously the expression of many proteins like cell specific markers and indicators of
apoptosis. Before starting your experiments it is important to note that apoptosis is not a static
process and certain tests are time specific. For instance, if most of your cells are entering into
late apoptosis you may not be able to detect early stages of apoptosis with some reagents. The
powerful technique of flow-cytometry can be used to both detect and quantify the level of
apoptosis in a population of cells at static points or in a time course.

SCHEMATIC DIAGRAM:

Working of Flow-Cytometry
 Flow-Cytometry analysis by different software

Application of Flow-Cytometry with respect to Apoptosis:

• Flow-cytometry is one of the most popular and versatile applications for studying
apoptosis.
• Two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow-
cytometry on the basis of differences in morphological, biochemical and molecular
changes occurring in the dying cells.
• One of the most common features of apoptosis that can be measured by flow-cytometry is
externalization of phosphatidylserine (PS), a phospholipid found in the inner membrane
of healthy cells.
• The powerful technique of flow-cytometry can be used to both detect and quantify the
level of apoptosis in a population of cells at static points or in a time course.
• Flow-cytometry allows the study of all aspects of apoptosis from induction via surface
receptors, to late stages where DNA fragmentation occurs.

CONCLUSION:

Cell death/ Apoptosis studies using Flow-Cytometry was done theoretically.