INTERNSHIP REPORT MIC 400:

Industrial/Research Organization
Attachment

Submitted by:JakiaRahmanJuthi
Student Id: 13326016
Program: BSc in Microbiology
Department: Mathematics and Natural Sciences
Date: 23 March, 2017
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This report entitled ‘Report on Internship’ at ‘ideSHi’ and ‘icddr,b’ is submitted by Jakia
Rahman Juthi for the requirement of the course of “MIC 400: Industrial / Research Organization
Attachment” , Microbiology program, Department of Mathematics and Natural Sciences, BRAC
University, Dhaka. The upcoming slides with paramount information about the experience and
work conducted throughout the course contain reference from various online data and protocols
provided by the aforementioned institutions.

Acknowledgement:
I want to express my deepest gratitude and special thanks to the chairperson of department of
Mathematics and natural sciences, BRAC University Professor A.A.Z. Ahmad, the chairman of
Bangladesh Atomic Energy Regulatory Authority Professor NaiyyumChoudhury and current
coordinator of Microbiology program Dr. MahboobHossain for providing me the opportunity to
understand, work and observe the respective laboratories.

I wish to express and claim my sincere, honest and deep gratitude to the Director of Centre for
Vaccine Science, Senior Scientist and Head of the Immunology Laboratory and Enteric Vaccine
Reasearch Group at icddr,b, and Executive Director, ideSHiDr. Firdausi Qadri for providing
me an opportunity to do my internship at the ‘ideSHi and icddr,b laboratories. I also want to
express my thanks to Dr. YasminAra Begum, Senior Scientist and officer of the Immunology
laboratory aticddr,b. I am very thankful to Dr.MD. KaiissarMannoor ( Scientist, Institute
development Science and health initiatives ), Dr. MD TaufiqurRahman ( Associate Scientist,
department of Enteric Vaccines) , Mr. Suprovath Kumar Sarkar – research fellow ( ideSHi) ,
Mr. GolamSarowerBhuyan – research officer ( ideSHi) and other staff and members at icddr,b
and ideSHi for allowing me to work there , giving necessary advices and guidance and arranging
all facilities to make the internship period easier. Without their support and kindness, I would not
have been able to complete my internship Course. I also want to thank all the employees of
ideSHi and icddr,b lab for their help which were extremely valuable for understanding all topics
both theoretically and practically.

Signature of the coordinator of Microbiology program

Department of Mathematics and Natural Sciences

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 Contents
1. Introduction……………………………………………………………………… 04
2. Organizations :

 Icddr,b………………………………………………….. 05
 ideSHi…………………………………………………….06

ideSHi laboratory:
3.Biosafety Training…………………………………………………………………..08
4.DNA isolation from human blood sample………………………………………....12
5. Detection of amplify a particular sequence of the DNA extracted
from human blood using polymerase chain reaction……………………………….16
6. Gel electrophoresis………………………………………………………………….18
7. Typhoid paratyphoid test ( TP test)………………………………………………..21
8.Ficoll Gradient Centrifugation Method and
antibodies from lymphocyte secretions (ALS)………………………………………..24
9. Gram staining………………………………………………………………………..27

Icddr,b laboratory:
12.Identification and confirmation of Vibrio cholera by dark field microscopy
and agglutination using monoclonal antibody(mAb)…………………………………32
13.Specimen culture on different agar plates(MacConkey, TTGA , SS )
for the isolation of E.coli and Vibrio cholerae………………………………………….34
14. ELISA:

 Coating for toxin ELISA………………………………………………………..41

 Toxin ELISA to detect LT/ST expression……………………………………...42
15.Identification of colonizing factors using Immuno Dot blot technique……………….45
16.Antibiogram to detect sensitivity of different antibiotics……………………………...49
17.Shigella Salmonella detection by biochemical test……………………………………..52
18.Abbreviation and Conclusion…………………………………………………………...59
19. References………………………………………………………………………….........60

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INTRODUCTION:
This Internship report covers the apprenticeship at the Institution for Developing Science and
Health Initiatives ( ideSHi) during 14 December, 2016 to 29 December, 2016. A group of
students were sent to icddr,b at Immunology department located on the 7 th floor and ideSHi
laboratory which is located on the second floor of institute of public health building in
mohakhali on the purpose of internship. The goal of icddr,b and ideSHi are to strengthen
scientific capacity and encourage innovations to make Bangladesh a local leader in the field of
health and multidisciplinary research globally. icddr,b focus on diarrhoealdisease control,
maternal and child health, nutrition and population sciences.Moreover, ideSHi focuses on
development of local scientists and scientific capacity, scientific research, public health
initiatives and educational programs. All the laboratory assistants were very humble and their
hospitality encouraged students to learn more about the equipments and scientific analysis done
in the laboratory. This internship report covers the apprenticeship at icddr,b and ideSHi during
the time period.

Competency Attainments:
The lab experience had an extremely steady part to learn distinctive techniques of Immunology,
molecular biology, study genomics and microbiological work procedure. During the internship, I
had the opportunity to actively learn, observe and practical working experience in some cases
that I have obtained during my education, such as the important aspects of laboratory techniques
and principles of the various methods. From icddr,b ( immunology department) I have observed
and experienced about Biosafety, Identification and confirmation of Vibrio choleraeby dark field
microscopy and agglutination using monoclonal antibody (mAB), Specimen’s culture on
different agar plates ( MacConkey, TTGA , SS) for isolation of E.coli and Vibrio cholera , DNA
template preparation from clture plate, detection of EnterotoxigenicEschrerichia coli ( ETEC)
toxin using PCR, coating for toxin ELISA and toxin ELISA to detect LT/ ST expression, dot blot
technique , Antibiogram. Furthermore, from ideSHi I have observed and gathered knowledge
about DNA extraction from blood, ELISA, TP test, PCR amplification , DNA extraction by
boiling method, gram staining.

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The International Centre for Diarrhoeal Disease Research, Bangladesh(icddr,b) is an
international health research organization located in Dhaka, Bangladesh. Dedicated to saving
lives through research and treatment, icddr,b addresses some of the most critical health concerns
facing the world today, ranging from improving neonatal survival to HIV/AIDS. In collaboration
with academic and research institutions over the world, icddr,b conducts research, training and
extension activities, as well as programmed-based activities, to develop and share knowledge for
global lifesaving solutions.From its origins as a center specializing in diarrheal diseases, it is now
an internationally recognized center of excellence across a wide range of conditions. icddr,b’s
work is guided by a number of key principles:

 High-quality science
 Local relevance
 Policy-oriented research
 Global networking
 Regional and global impact
 Training and capacity-building

icddr,b provide laboratories with well equipped to carry out research based on the latest
technologies. They have developed laboratory facilities to support a wide range of
microbiological, immunological, genetic and other research. These facilities underpin our
laboratory studies on disease-causing organisms and host immune responses to them.So, scientific
expertise and research infrastructures enable us to address key health issues from multiple
directions and to carry out the interdisciplinary studies that will be required to address the most
intractable health challenges that Bangladesh and other developing countries are now facing.

One of our important aims is to integrate field-based surveillance and intervention studies with
detailed molecular and cellular analyses at our central laboratory facilities.

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The incentive for establishing “Institution for developing Science and Health Initiatives
(ideSHi)” was born when the Christophe Rodolfe Grand prize was awarded to Firdausi Qaderi in
June 2012. A seven member executive board was formed with experts and entrepreneurs to plan
and formally register ideSHi in Bangladesh as a nonprofit foundation. It was felt that Bangladesh
needs to meet the health challenges in our settings by carrying out sophisticated biomedical
training and capacity building as well as innovative research. The research institute is now
located in the premises of the Centre For Medical Biotechnology of the Directorate General of
Health Services, Institute of public health in Dhaka. It took about 3 months to renovate the space,
procure and install equipment and set up the ideSHi facilities to meet BSL -2 requirements to
carry out molecular, immunological and genetics studies as well as hands –on training in the
field of biomedical science and biotechnology. The institute is not merely confined to newborn
screening but it will also helpful for molecular diagnosis of the hereditary disorders that starts in
later ages of life, and during pregnancy where genetic disease management and genetic
counseling are needed. ideSHi also plans to serve as the hub for innovative advanced genomics
by carrying out research and training activities in this field.

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Genomic and Microbiology Unit:

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Introduction and Biosafety training:

Purpose:The main purpose of this topic is to introduce the laboratory and training regarding
the Biosafety which are maintain into the laboratory.

A biosafety level is a set of bio-containment precautions required to isolate dangerous biological

agents in an enclosed laboratory facility. The levels of containment range from the lowest
biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4).A fundamental objective of any
biosafety program is the containment of potentially harmful biological agents. The term
“containment” is used in describing safe methods, facilities and equipment for managing
infectious materials in the laboratory environment where they are being handled or maintained.
The purpose of containment is to reduce or eliminate exposure of laboratory workers, other
persons, and the outside environment to potentially hazardous agents. The use of vaccines may
provide an increased level of personal protection. The risk assessment of the work to be done
with a specific agent will determine the appropriate combination of these elements.

At the lowest level of biosafety, precautions may consist of regular hand-washing and minimal
protective equipment. At higher biosafety levels, precautions may include airflow systems,
multiple containment rooms, sealed containers, positive pressure personnel suits, established
protocols for all procedures, extensive personnel training, and high levels of security to control
access to the facilities.

ideSHi and icddr,b follows universal precaution during work which is “All samples are
infectious”. They always maintain good laboratory practice. Biosafety consists of 4 levels which
are:

Biosafety Level 1:

Associated with non-infectious agent. Biosafety level 1 is suitable for working withwell
characterized agents which do not cause disease in healthy humans. At this level, precautions
are limited relative to other levels. Work can be done in open bench.

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Biosafety level 2:
Biosafety Level 2 practices, equipment, and facility design and construction are applicable to
clinical, diagnostic, teaching, and other laboratories in which work is done with the broad
spectrum of indigenous moderate-risk agents that are present in the community and associated
with human disease of varying severity. With good microbiological techniques, these agents can
be used safely in activities conducted on the open bench, provided the potential for producing
splashes or aerosols is low. Hepatitis B virus, HIV, the Salmonella, and Toxoplasma and
organisms that belong to risk group 2 are assigned to this containment level. BSL-2 is
appropriate when work is done with any human-derived blood, body fluids, tissues, or primary
human cell lines where the presence of an infectious agent may be unknown. (Laboratory
personnel working with human derived materials should refer to the OSHA Blood borne
Pathogen Standard2 for specific required precautions).

Primary hazards to personnel working with these agents relate to accidental percutaneous or
mucous membrane exposures, or ingestion of infectious materials. Extreme caution should be
taken with contaminated needles or sharp instruments. Even though organisms routinely
manipulated at BSL-2 are not known to be transmissible by the aerosol route, procedures with
aerosol or high splash potential that may increase the risk of such personnel exposure must be
conducted in primary containment equipment, or in devices such as a BSC or safety centrifuge
cups. Personal protective equipment should be used as appropriate, such as splash shields, face
protection, gowns, and gloves.

Secondary barriers, such as hand washing sinks and waste decontamination facilities, must be
available to reduce potential environmental contamination.

Laminar flow

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Biosafety Level 3:

Biosafety Level 3 practices, safety equipment, and facility design and construction are applicable
to clinical, diagnostic, teaching, research, or production facilities in which work is done with
indigenous or exotic agents with a potential for respiratory transmission, and which may cause
serious and potentially lethal infection. Work on Highly infectious organisms and airborne
organisms should be performed in lab where bio safety level 3 is maintained. Mycobacterium
tuberculosis, St. Louis encephalitis virus, and Coxiellaburnetiiand organisms that belong to risk
group 3 are assigned to this level. Primary hazards to personnel working with these agents relate
to autoinoculation, ingestion, and exposure to infectious aerosols.

At BSL-3, more emphasis is placed on primary and secondary barriers to protect personnel in
contiguous areas, the community, and the environment from exposure to potentially infectious
aerosols. For example, all laboratory manipulations should be performed in a BSC or other
enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this
level include controlled access to the laboratory and ventilation requirements that minimize the
release of infectious aerosols from the laboratory

Biosafety Level 4:

Biosafety level 4 is the highest level of biosafety precautions, and is appropriate for work with
agents that could easily be aerosol-transmitted within the laboratory and cause severe to fatal
disease in humans for which there are no available vaccines or treatments. It is needed in
extremely hazardous condition. Air lock, shower entry must be present. Such as, HIV / Ebola
related research.

Each level is associated with appropriate Equipment, practices and work procedures. Diagnostic
and health-care laboratories must follow biosafety level 2 or above. Icddr,b and ideSHi maintains
Biosafety level 2 and 3 according to requirements.

Their standard characteristics include:

1. No food inside lab

2. No protective cloths or else outside the lab

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3. Clean bench after work

4. At first autoclave then incineration must be done after discard

5. 70% alcohol should be used

6. Autoclave temperature is 1210c at 15 psi. Autoclave success is determined by autoclave tap or

culturing sample after autoclave.

7. Sample must be received in the transportation room.

Figure: A glance of Biosafety measures present in ideSHi and icddr,b

Immunology laborator

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DNA isolation from human blood :

Purpose:
Extraction of genomic DNA from blood for genetic analysis and observation at the genomic
level.

Principle:
Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various sources.
Methods used to isolate DNA are dependent on the source, age, and size of the sample. Despite
the wide variety of methods used, there are some similarities among them. In general, they aim
to separate DNA present in the nucleus of the cell from other cellular components. Presence of
proteins, lipids, polysaccharides and some other organic or inorganic compounds in the DNA
preparation can interfere with DNA analysis methods, especially with polymerase chain reaction
(PCR). They can also reduce the quality of DNA leading to its shorter storage life.

The isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This process is
essential for the destruction of protein structures and allows for release of nucleic acids from the
nucleus. Lysis is carried out in a salt solution, containing detergents to denature proteins or
proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both. It results in
the breakdown of cells and dissolving of membranes.

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 Figure: DNA isolation from human blood.

DNA isolation is a process of purification of DNA from sample using a combination of physical
and chemical methods. For the chemical method, there are many different kits are used for
extraction, and selecting the correct one will save time on kit optimization and extraction
procedures. DNA isolation is a procedure that can result in high amount of DNA isolation from
human blood in several steps using chloroform as one of the main components. The WBCs are
the targeted cells from which the DNA is isolated as they are usually the most convenient source
of human genomic DNA. The steps of the procedure are sequentially given below along with the
chemicals needed:

Standard chemicals:
 EDTA
 1M Tris- HCl
 Red blood cell lysis buffer
 Nucleic lysis buffer
 TE buffer
 Chloroform

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  Ethanol.

Procedure
1. Five hundred µl of blood was poured into a 1.5 ml eppendorf tube and 1000 µl of red cell
lysis buffer was added.

2. The tube was shaken gently up to homogenizing then it was centrifuged for 2 minutes at 7000
rpm.

3. Supernatant was discarded and steps were repeated 1-3 two or three more times to remove
hemoglobin.( Important to break down the pellet by vortexing and it was rinsed well in order to
clean white blood cells from residual of hemoglobin)

4. The tube was placed on tissue paper for few seconds downward. (careful from cross
contamination between different samples)

5. Four hundred micro-litter of nucleic acid lysis buffer was added to eppendorf tube. It was
vortexed or pippetted slowly.( if pellet formed must be pipetted up to dissolve it or if foam forms
should be centrifuged)

6. One hundred µl of saturated NaCl(5m) was added and then 600 µl of chloroform was added
to eppendorf tube and mixed on a rotating blood mixer at room temperature.

7. Then it was centrifuged for 2 minutes at 7000 rpm.

8. After that 400 µl of supernatant was transferred to a new 1.5 ml tube. 9. 800 µl of cold
absolute ethanol (-20°C) was added and it was shaken gently.

10. Then it was vortex (DNA should appear as mucus like strand in the solution phase)

11. The tube was then centrifuged for 1 minute and 12000 rpm to precipitate.

12. The supernatant was discarded carefully and the tube is dried completely in room
temperature. (eppendorf tube was placed downward on the tissue paper)

13. Fifty µl of TE buffer (nuclease free water) was added to it and then vortexed.

14. Eppendorf tube of DNA was then kept in 4°C – 20 °C for later uses.

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Results:
DNA was successfully purified from blood.

Figure: DNA isolation from human blood

DNA isolation is a very sensitive process. It should be handled very carefully. The presence of
DNA can be confirmed by gel electrophoresis.

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Polymerase Chain Reaction (PCR):
Purpose:
To amplify a particular sequence of the DNA extracted from human blood.

Principle:
The polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a
single copy or a few copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence. PCR is a rapid, inexpensive and
simple way of copying specific DNA fragments from minute quantities of source DNA material,
even when that source DNA is of relatively poor quality. DNA exists as a double-stranded
molecule. Some areas of DNA contain genes while others control gene expression. In DNA one
strand is the coding strand and the other is the non-coding strand. The coding strand is the non
template strand. The non-coding strand is actually the template which is used for PCR to amplify
DNA.

The host strain choice for your template preparation is an important consideration, as some
strains will yield higher quality DNA for downstream applications. Some host strains release
certain factors during lysis (endonucleases, carbohydrates, etc.) that can degrade DNA or inhibit
the sequencing reaction.

The amount of total DNA in a PCR has a marked effect on the outcome of a PCR procedure.
Using too much total DNA results in packed DNA in the confined space of the reaction vessel
and can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of
large Taq polymerase molecules. However the ratio of target DNA to burden DNA is also
important. The concentration of the target DNA should be balanced with the number of cycles in
the reaction.

Materials:
Buffers and Solutions: 10x Amplification buffer Chloroform dNTP solution (20 mM) containing
all four dNTPs (pH 8.0) Enzymes and Buffers: Thermostable DNA polymerase NucleicAcids
and Oligonucleotides Forward primer (20 μM) in H2O Reverse primer (20 μM) in H2O
Template DNA. Master Mix:

PCR buffer = 1×3 = 3 µl

MgCl2 = 0.2×3 = 0.6 µl

dNTPs = 1.6×3 = 4.8 µl

Q solution = 2×3 = 6 µl

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Hot start = 0.05×3 = 0.15 µl

Primer (Forward and Reverse) = 0.4×3 = 1.2 µl

Water = 3.75×3 = 11.25 µl

DNA = 1 µl

The Thermal Cycle:

1. 950 C for 5 minutes (initial denaturation).

2. 940 Cfor 30 seconds for denaturation.

3. 54 0 C for 30 seconds for primer Annealing.

4. 720 C for elongation for 30 seconds.

5. 720 C for elongation for 5 minutes. Storage at 40 C until further use.

Procedure:
1. To run, the comb was gently removed; tray was placed in electrophoresis chamber, and
covered (just until wells are submerged) with electrophoresis buffer (TBE buffer, the same buffer
used to prepare the agarose).
2. To prepare samples for electrophoresis, 1.5μl of gel loading dye was added for every 1μl of
DNA solution.

3. The PCR Master Mix was loaded along the dye.

4. The gel was run at 60 V which took approximately 1 hour for the run to be complete.

5. The gel was stained in 0.5 μg/ml ethidium bromide until the DNA has taken up the dye
and was visible under short wave UV light.

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Gel Electrophoresis:
Purpose:
Gel electrophoresis is used to separate macromolecules like DNA, RNA and Proteins. DNA
fragments are separated according to their size. Proteins can be separated according to their size
and their charges.

Principle:
Agarose gel electrophoresis is the easiest and most popular way of separating and analyzing
DNA. Here DNA molecules are separated on the basis of charge by applying an electric field to
the electrophoretic apparatus. Shorter molecules migrate more easily and move faster than longer
molecules through the pores of the gel and this process is called sieving. The gel might be used
to look at the DNA in order to quantify it or to isolate a particular band. The DNA can be
visualized in the gel by the addition of ethidium bromide. Agarose is a polysaccharide obtained
from the red algae Porphyraumbilicalis. Its systematic name is (1 4)-3, 6-anhydro-aL-
galactopyranosyl-(1 3)-β-D-galactopyranan. Agarose makes an inert matrix. Most agarose gels
are made between 0.7% and 2% of agarose. A 0.7% gel will show good separation for large
DNA fragments (5-10kb) and a 2% gel will show good resolution for small fragments with size
range of 0.2-1kb. Low percentage gels are very weak (Note: it may break when you lift them)
but high percentage gels are usually brittle and do not set evenly. The volume of agarose
required for a mini-gel preparation is around 30-50ml and for a larger gel, it is around 250ml.

Structure of agarose:

Factors affecting the movement of DNA:

 Voltage Applied: The migration rate of the linear DNA fragments through agarose gel is
proportional to the voltage applied to the system. As voltage increases, the speed of DNA
also increases. But voltage should be limited because it heats and finally causes the gel to
melt.

 EthidiumBromide (EtBr): It is an intercalating agent which intercalates between nucleic

acid bases and allows the convenient detection of DNA fragments in gel. When exposed
to UV light, it will fluoresce with an orange colour. After the running of DNA through an
EtBr-treated gel, any band containing more than ~20 ng DNA becomes distinctly visible
under UV light. EtBr is a known "mutagen", however, safer alternatives are available. It
can be incorporated with agarose gels or DNA samples before loading, for visualization
of the fragments. Binding of Ethidium bromide to DNA alters its mass and rigidity, and
thereby its mobility. Buffers: Several different buffers have been recommended for

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 electrophoresis of DNA. The most commonly used buffers are Tris-acetate-EDTA (TAE)
and Tris-borate-EDTA (TBE). The migration rate of DNA fragments in both of these
buffers is somewhat different due to the differences in ionic strength. These buffers
provide the ions for supporting conductivity. Conformation of DNA: DNA with different
conformations that has not been cut with a restriction enzyme will migrate with different
speeds. Nicked or open circular DNA will move slowly than linear and super coiled DNA
(slowest to fastest: nicked or open circular, linear, or super coiled plasmid). Super helical
circular, nicked circular and linear DNAs migrates gels at different rates through agarose
gel. The relative nobilities of these three forms depend on the concentration, type of
agarose used to make the gel, applied voltage, buffer, and the density of super helical
twists.

Procedure:
1. Fifty milliliter TBE buffer and 0.5 gm agarose were added in a beaker and heat was applied.

2. When agarose was dissolved in buffer, it was then kept to cool down.

3. After that 1 µl gel red was added in the beaker and mixed.

4. Appropriate comb for sample slots in gel was chosen and gel was poured in the mold.

5. When the gel was settled down, comb was removed carefully.

6. Then in each well 3µl PCR product and 2 µl loading dye was added and in one well 4 µl
ladder was added.

7. Then the gel was kept on the gel electrophoresis tank.

8. Electrophoresis was done for 30-40 minutes at 150 volt.

9. Then the gel was loaded onto the gel dock and the results were analyzed.

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Results:
Observation of DNA bands of the sample as the isolation of DNA sample was done properly.

Figure:DNA bands in Gel Electrophoresis

Interpretation of results:
Agarose gel electrophoresis is an effective means of determining if a restriction digest procedure
has been successful. DNA bands had been observed under UV light. Bands are separated by their
size and molecular weight. All the lengths of DNA bands are equal. Although agarose gel
electrophoresis can be used for a variety of purposes, two of the most common uses with respect
to DNA are verifying the success of a restriction digest or a ligation step in an experiment. Due
to occurrence of handling error we didn’t get the desired result for this particular experiment.

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Typhoid paratyphoid test(TP Test):
Purpose:
TP/TPT test is typhoid or para-typhoid test. The main purpose of this test is to see the response
of antibody against typhoid or para-typhoid antigen in patient’s blood sample.

Principle:
TP Test detects Salmonella-specific IgA responses in lymphocyte culture supernatant. TP Test is
done in patients with suspected enteric fever, patients with other illnesses, and healthy controls.
The immunology unit at ideSHi works for the diagnosis of immunological disorders and
infectious diseases. One of the main diagnostic tests conducted by this unit is TP (Typhi and
Paratyphi test).

Salmonella enterica serotype Typhi and Paratyphi A are responsible for typhoid and paratyphoid
fever respectively and the disease caused is known generally as enteric fever. Appropriate and
early diagnosis of the disease is important for initiation of treatment of the patient with a suitable
antibiotic. The performance of the available diagnostic methods takes time and as well as have
high sensitivity and specificity. A kinetic ELISA based method is used in this test since it is fast,
highly sensitive and requires maximum 48 hours to identify the antibody against pathogen. It is
an immunodiagnostic assay, the TP Test, which is based on the use of secretions of antibodies
from peripheral blood lymphocytes. The simplifications of the procedure shows that the assay
has as a sensitivity of 100% for detecting patients with typhoid and paratyphoid fever in an
enteric fever endemic zone. The TP Test uses a small blood volume, and reverts to negative by
convalescence, supporting its further development as a relatively low cost assay to diagnose
patients with enteric fever in endemic zones.

Materials used:
 Ninety six well ELISA plates
 Pipettes and multichannel pipettes

Reagents:
 PBS (phosphate buffer saline): For washing and diluting samples
 BSA (bovine serum albumin): For blocking unbound region of the plates. The plates
are blocked at empty sites without bound antigens so that the antibodies do not stick to
the surfaces in those places. This might happen as both antigens and antibodies are
protein in nature. This might give false positive result with secondary antibodies bind to
the non specifically bound primary antibodies.

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  Pool: A mixture of samples from several known typhoid patients and made into
250mAb/min at 450nm. So the antigens are known to be present in them.
 Diluent: 0.1% BSA PBS
 Primary antibody: Rabbit anti-human IgA
 Secondary antibody: Anti rabbit antihuman IgA with HRP conjugate.
 Substrate: 10 ml sodium citrate mixed with 0.01gm OPD (ortho phenyl diamine) and 4
micro liter H 2O 2.

Procedure:
1. Plate Coating: The ELISA plates was coated with MP (membrane preparation) antigen by
adding 100µl of antigen per well. The plates were then kept for overnight incubation at room
temperature.

2. PBS wash: After overnight incubation the ELISA plates were washed 3 times with 1% PBS
and shaken swiftly to remove the liquids from the wells.

3. Blocking agent addition: 200µl of BSA-PBS (Bovine serum albumin-phosphate buffer

saline) tween was added to each well of the plates. This blocking agent blocks the unbound well
surface with non-human protein. The plates were then incubated at 37ºC for 30 minutes.

4. Wash: The ELISA plates were first washed 3 times with PBS-Tween at 1 minute intervals.
After that plates were washed with PBS.

5. Negative Control Addition: 100µl of 0.1% BSA-PBS Tween was added as negative control
in 3 wells (2nd, 3rd and 4th) of the ELISA plates.

6. Positive Control Preparation and Addition: 1:100 dilution of pool plasma was created by
using 0.1% BSA-PBS Tween as diluent. 100µl 0.1% BSA-PBS Tween was added for every 1µl
of pool and pool plasma preparation. 100µl each of this pool plasma was added as positive
contron in 3 wells (6th,7th and 8th) of the ELISA plate.

7. Sample Addition: 50µl of 0.1% BSA-PBS Tween followed by 50µl of ALS (Antibody in
Lymphocyte Secretion) sample of patient was added in the 10th well of the ELISA plate.

8. Incubation: The ELISA plates were incubated at 37ºC for 1hour 30 minutes. 9. Wash: After
completion of incubation, the ELISA plates were first washed 3 times with PBS-Tween at 1
minute’s intervals. After that plates are washed with PBS.

10. Conjugate preparation and addition: 1:1000 dilution of Anti-Human IgA HRP is created
by using 0.1% BSA-PBS Tween as diluent. 100µl of 0.1% BSA-PBS Tween is added for every
1µl of Anti-Human IgA HRP, Mixed by Vortex and conjugate is prepared. 100µl of each of this
conjugate was added to the wells of the ELISA plate.

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11. Incubation: The ELISA plates are incubated at 37ºC for 1 hour 20 minutes.

12. Wash:After completion of incubation, the ELISA plates are first washed 3 times with PBS-
Tween at 1 minute intervals. After that plates are washed with PBS.

13. Substrate Addition: The substrate for Horse Radish Peroxidase (HRP) is
ophenylenediamine (OPD). OPD solution is prepared by adding 0.01g of OPD to 10ml sodium
citrate solution. 4µl H2O2 is added to this OPD solution. 100µl of this substrate solution is added
to each well of the ELISA plate.

Figure: After adding substrate

14. Reading: The readings of the plates are taken at ELISA reader for measuring the colour
intensity. The reading is compared with the pool value and expressed in percentage.

Interpretation of Results:
Readings were taken from ELISA plate reader at an interval of 19 seconds. The optical density
of the plasma pool or positive control lies between 250-380, was considered and its average was
calculated. Now the reading for individual sample, which was 2X diluted, was multiplied by 2.
This value was then divided by the calculated average value of the pool. Then negative result
came out which indicate that the patient was not infected because the reading shows less than 10
average absorbance values which was 4.270463. If it is more than 10 the result should be
considered positive.

23
Ficoll Gradient Centrifugation Method And Antibodies
From Lymphocyte Secretions:
Purpose:
This technique is used to prepare ALS (Antibody in Lymphocyte Secretion) which is used as a
sample in the TP test.

Principle:
The TP test procedure has already been discussed before. However, an essential task required to
carry out this TP test in MNC collection. The ALS which is used as sample in the TP test is
prepared by MNS.

Figure: MNC isolation.

24
MNC isolation can be done by two methods:

1.Ficoll gradient centrifugation

2. RBC lysis

Isolation of prepheral blood mononuclear cell (PBMC) by ficoll gradient centrigufation:

Ficoll is a polysaccharide with a density of 1.077g/ml. It has higher density than MNC but
lower density than RBC. The procedure for isolation of MNC using ficoll is given below:

1. Blood was collected from patients by venipuncture procedure and placed in a sterile
heparinized tube to avoid coagulation.

2. Two milliliter of heparinized venous blood was taken in a falcon tube and diluted with same
volume of PBS.

3. The diluted blood was gently added onto the ficoll, which has a slightly less volume than
blood, in order to maintain two distinct layers.

4. The tube was centrifuged at 772g for 25 minutes at 20-25ºC in the Sorvall RT 6000 B
Refrigerated Centrifuge. After completion of centrifugation, four layers formed, which are from
the top: Plasma, MNC, ficoll and RBC.

5. The plasma was transferred into a labeled eppendorf and stored for plasma ELISA and
vibriocidal tests.

6. The cloudy layer of MNCs were removed carefully from the top of the ficoll layer with a
pipette and collected in a falcon tube. The RBCs are discarded.

7. PBS added to the falcon tube containing MNCs, to make the volume about 15ml. the tube is
centrifuged at 953g for 10 minutes at 20-25ºC.

8. The supernatant is discarded upon completion of incubation and the pellet containing cells are
re-suspended in 10ml of PBS.

9. The cells are counted in 25 large squares of the hemocytometer, by mixing 25µl of the cells
with 25µl trypan blue.

10. The falcon tube was again centrifuged at 953g for 10 minutes at 4ºC.

11. The total number of cells in 10ml was calculated as: cells suspension dilute with trypan blue
ratio is about 1:2 and total numbers of cells are counted by the help of hemocytometer.

25
12. After completion of centrifugation, the supernatant was discarded. The cells resuspended in
RPMI complete medium so that for each million cells 100µl of RPMI media is required. For
example, 1ml media is required for 10 million cells.

Figure: In the first picture, Ficoll was added and then centrifuged
and as a result, getting four layers which was showed in the second
picture.

26
Gram staining:
Purpose:

Gram stain is probably one of the most commonly used staining procedures used in the field of
microbiology. It is one of the differential stains that are used to characterize bacteria in one of
two groups: either gram positive bacteria or gram negative bacteria.

Principle:

Gram Staining is the common, important, and most used differential staining techniques in
microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884.
This test differentiates the bacteria into Gram Positive and Gram Negative Bacteria, which helps
in the classification and differentiations of microorganisms.

Gram staining is a common technique used to differentiate two large groups of bacteria based on
their different cell wall constituents. The Gram stain procedure distinguishes between Gram
positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria
stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains
the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red,
which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during
the decoloring process.

Figure: Bacterial staining method.

27
When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of
the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell
walls of gram positive bacteria have a thick layer of protein-sugar complexes called
peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to
dehydrate and shrink which closes the pores in the cell wall and prevents the stain from exiting
the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the
thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in color.

In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the
thin layer of peptidoglycan and thick outer layer which are formed of lipids, CV-Iodine complex
gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell
walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again
stained with safranin, they take the stain and appear red in color.

Materials used:

 Crystal Violet, the primary stain

 Iodine, the mordant
 A decolorizer made of acetone and alcohol (95%)
 Safranin, the counterstain  Microscopic Slides

Procedure:

1. Two individual sterile glass slides were taken onto which an appropriate amount of normal
saline was taken.

2. A single colony from the culture plate of S. pneumoniae and K. pneumoniae were taken with
the sterile loops and mixed with the saline on the slides.

3. The slides were air dried.

4. The films were fixed on the slide by passing them through the Bunsen burner two or three
times without exposing the dried film directly to the flame.

28
5. The slides were flooded with crystal violet solution for up to one minute. After one minute, the
slides were washed off with distilled water and drained.

6. The slides were then flooded with Gram’s Iodine solution and allowed to act for one minute,
then washed off and drained.

7. The slides were again flooded with acetone and washed off instantly with water. The slides
were then drained.

8. Next the slides were flooded with safranine and kept for one minute. Then they were washed
off and drained.

9. The glass slides were dried next and observed under microscope.

Results:

Figure: Gram Positive cells Figure: Gram Negative cells

Violet diplococci and pink diplococci cells were observed individually. This means both gram
positive and gram negative cells were present.

29
Interpretation of results:

As the bacterial cultures used in this experiment were S. pneumoniae and K. pneumoniae and
which was already known. So the main purpose of this experiment was to identifying and
ensuring that the cultures were either Gram positive or Gram negative. As Gram positive shows
purple or violet color colony and Gram negative shows pink/red color colony so according to the
observed result it was concluded that S.pneumoniae belongs to Gram positive and K.
pneumoniae belongs to Gram negative family.

30
Immunology Unit :

31
Identification and confirmation of Vibrio cholerae by dark
field microscopy and agglutination using monoclonal
antibody ( mAB):
Purpose of the experiment: To observe the “shooting star” motility of the bacteria and
confirmation by the inhibition with mAbs.

Principle:The examination of diarrheal stool of identity the presence of V. cholerae is usually

done by dark field microscope. The motility of bacteria is sensitive with the time. In a dark field
microscope a sample is placed on the stage it appears bright against a dark background. As there
is no media on the glass slide the mobility would be seen less as the time passed. Cholera
enterotoxin has two sites: A for action slide and B for binding slide. Three types of antigen are
there: Ogawa, Inaba and 0139. These are monovalent antigens. The procedure was done to detect
motile V. cholera in stool. This is a method which excludes the unscattered beam from the
image. As a result, the field around the specimen is generally dark. The confirmation was done
by using three types of antisera (monoclonal antibody) were used. For inhibition of V. cholera
01- Ogawa, V. cholerae 01- Inaba, or V. cholerae0139 specific monoclonal antibodies (MAB)
were used. The specific MAb showed the inhibition against sero types for the presence of
specific antigen (Ags). The inhibition is observed by clump formation or the slide and under the
dark field microscope with inhibition of the motility. The non-specific types (antigen) against
mAb still showed least motility.

Figure: V. cholera( just like star)was identified by using dark field

microscope

Procedure:
1. A microscopy slide was taken divided into three parts.

32
 2. After that,10 μL of the sample was taken in three parts.
3. Three types of antisera (- Ogawa, V. cholerae 01- Inaba, or V. cholerae0139 specific
mAb ) was applied on the slide and cover slip was applied too.
4. Placed under the dark field microscope of 10x -40x magnifications.
5. Pigmented motile object (bacteria) was seen.

Observation and Result:The following results were seen during the experiment.
Types of antisera 01 - Ogawa 1- Inaba 0139
(mAb)
Motility Seen Not seen Not seen ( least)

Interpretation:

If the coagulation was occurred then it was proved that a patient will suffer from the cholera if
not then a patient is healthy.

33
Specimen culture on different agar plates for the isolation of
E.coli and V.cholerae:
Purpose: Purpose is to detect E.coli and V.choleraeon different agar plates.

Mac Conkey agar:

MacConkey agar is used for the isolation of gram-negative enteric bacteria and the
differentiation of lactose fermenting from lactose non-fermenting gram-negative
bacteria. Pancreatic digest of gelatinand peptones (meat and casein) provide the essential
nutrients, vitamins and nitrogenous factors required for growth of
microorganisms. Lactosemonohydrate is the fermentable source of carbohydrate. The crystal
violet and bile salts act as selective agent, which inhibit the growth of gram-positive
bacteria. Sodium chloride maintains the osmotic balance in the medium. Neutral red is a pH
indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Agar acts
as the solidifying agent.
Lactose fermenting organisms grow as red or pink on MacConkey agar and may be
surrounded by a zone of acid precipitated bile. The red color is due to production of acid from
lactose, absorption of neutral red and a subsequent color change of the dye when the pH of
medium falls below 6.8.

Lactose non fermenting organisms such as salmonella and shigella remain colorless and
typically do not alter the appearance of the medium.

Composition:

Ingredients Amount

Peptone (Pancreatic digest of gelatin) 17 gm

Proteose peptone (meat and casein) 3 gm

Lactose monohydrate 10 gm

34
Bile salts 1.5 gm

Ingredients Amount

Sodium chloride 5 gm

Neutral red 0.03 gm

Crystal Violet 0.001 g

Agar 13.5 gm

Distilled Water Add to make 1 Liter

Figure: MacConkey agar (E.coli)

35
TaurocholateTellurite Gelatin Agar (TTGA):
TTGA agar is used for selective isolation and differentiation of Vibrio cholerae and other Vibrio
species from pathological samples like feces or rectal swabs.Casein enzymatic hydro lysate in
the medium supplies essential nutrients. Sodium taurocholate inhibits the contaminating gram
positive bacteria. Potassium tellurite is a selective and differential agent. It inhibits many gram-
positive bacteria and due to the reduction reaction the colonies form a grey to black color.
Sodium chloride maintains the osmotic equilibrium while sodium carbonate helps in maintaining
the elevated pH of the medium. Gelatin acts as an additional carbon and energy source. The high
pH and potassium tellurite are inhibitor to most Enterobacteriaceae and gram-positive bacteria,
though Proteus may form grey centered colonies without a halo. Overnight growth of V. cholera
on TTGA agar, small opaque colonies with slightly dark centers is viewed. Further incubation
makes the centers of the colonies become darker and eventually the entire colony becomes
“gunmetal” grey in color. Additional tests like antisera and/or biochemical are needed to isolate
particular species from this medium.

Composition of TTGA agar:

Ingredients (Gms) Amount (Liter)

Casein enzymichydrolysate 10.000

Sodium chloride 10.000

Sodium taurocholate 5.000

Sodium carbonate- 1.000

Gelatin- 30.000

36
 Agar 15.000

Final pH (at 25°C) -8.5±0.25

Figure: colonies of Vibrio cholerae with dark spot and hallow zone on
TTGA media

Salmonella shigella (SS) agar:

Salmonella Shigella (SS) Agar is moderately selective and differential medium for the isolation,
cultivation and differentiation of Salmonella spp. and some strains of Shigella spp. The inclusion
of Bile Salts, Sodium Citrate and Brilliant Green serve to inhibit gram-positive, coliform
organisms and inhibit swarming Proteus spp., while allowing Salmonella spp. to grow.  Beef
Extract, Enzymatic Digest of Casein, and Enzymatic Digest of Animal Tissue provide
sources of nitrogen, carbon, and vitamins required for organism growth. Lactose is the
carbohydrate present in Salmonella Shigella Agar. Thiosulfate and Ferric Citrate permit
detection of hydrogen sulfide by the production of colonies with black centers. Neutral red turns
red in the presence of an acidic pH, thus showing fermentation has occurred.

37
Salmonella will not ferment lactose, but produce hydrogen sulfide (H 2S) gas.  The resulting
bacterial colonies will appear colorless with black centers. But salmonella typhi is late lactose
fermenting and produces less H2S. Salmonella paratyphi is non-lactose fermenting and doesn’t
produce H2S.Shigella do not ferment lactose or produce hydrogen sulfide gas, so the resulting
colonies will be colorless. Only Shigellasonnei is late lactose fermenting so it can grow in SS
agar.

Composition of SS Agar:

Ingredients Gms / Litre

Beef Extract 5.00

Enzymatic Digest of Casein 2.50

Enzymatic Digest of Animal Tissue 2.50

Lactose 10.00

Bile Salts 8.50

Sodium Citrate 8.50

Sodium Thiosulfate 8.50

Ferric Citrate 1.00

Brilliant Green 0.00033

Neutral Red 0.025

38
 Agar 13.50

Distilled Water = 1000 ml

pH ( at 25°C) 7.0 ± 0.2

Figure: SS agar (S.typhi)

Specimen culture and isolation:

Patient stool or rectal swab samples are collected and documented. To identify the
microorganisms in the sample, they are first cultured on different agar mediums. According to
the specific characteristics and tests those microorganisms are identified. Vibrio cholera
andEscherichia coli are the targeted organism of the tests.

Procedure:
1. Sample: Fresh Diarrheal Stool or Stool

2. Macroscopic Examination: Rice Watery appearance of fresh stool.

39
3. Collection : Fresh Diarrheal Stool or Stool is collected and should be collected on two
or three swabs/rectal swab and placed in a container with transport medium called Cary–
Blair

4. Culture :
 Sample was poured in a sampling tube and cotton tipped stick was used to take out the
sample.
 A thick smear was made by the cotton bud on the agar plate
 Streaking procedure was done on the agar plate from the smear and then incubated at
37°C overnight.

Figure: TTGA agar plate (subculture)

40
Toxin ELISA to detect LT/ST expression:
Purpose: To detect LT/ ST expression

Enzyme-linked immunosorbent assay (ELISA) test is the most widely used type of
immunoassay. ELISA is a rapid test used for detecting or quantifying antibody (Ab) or antigen
(Ag) against viruses, bacteria and other materials. ELISA is so named because the test technique
involves the use of an enzyme system and immunosorbent.

A general ELISA is a five-step procedure which includes coating the microliter plate wells with
antigen; blocking all unbound sites to prevent false positive results; adding primary
antibody (e.g. rabbit monoclonal antibody) to the wells; then adding secondary
antibody conjugated to an enzyme (e.g. anti-mouse IgG); and reaction of a substrate with the
enzyme to produce a colored product, thus indicating a positive reaction.

Different types of ELISA are used including direct, indirect, sandwich, competitive ELISA but
among them sandwich ELISA is most commonly used. Toxin ELISA that is used in icddr,b to
detect LT/ST toxin of ETEC also uses the principle of sandwich ELISA. In sandwich ELISA, the
ELISA plates are coated with captured antibody, then sample containing the antigens are added.
These antigens will bind to the captured antibody. After that secondary antibody conjugated with
enzyme is added which will bind to the sample containing the antigen? Then color is developed
by adding substrate. If color develops in LT plate of toxin ELISA then it is considered to be LT
positive. If no color develops in ST plate then it is considered to be ST positive.

41
Procedure:
LT plates:
1. E.coli isolates were cultured on Mac conkey agar plates. Differentiated streaks were used
so that at least 6 colonies can be identified. The plates were incubated at 37˚C overnight.
The plates can be stored at -4˚C for 1-2 weeks before testing in LT/ST Elisa
2. Elisa plates were coated with 100ul of ganglioside GM1 solution
3. After coating plates were kept overnight at room temperature. The plates can be stored at
-4˚C for less than or up to 2 weeks until used.
4. Then GM1 coated wells were washed 2-3 times with PBS buffer
5. The wells were then blocked by adding 200 µl of 0.1% BSA-PBS
6. Plates were incubated at 37˚C for 30 minutes
7. In the meantime LB broth+ linkomycin solution was prepared and vortexed. LB broth
helps selective bacteria to grow and linkomycin kills other bacteria which were not
needed
8. After blotting the plates were washed 1time with PBS
9. One hundred µl of LB broth+ linkomycin was added to each well of the plates
10. Bacteria inoculation was performed. For bacteria inoculation 6 colonies confirmed by
PCR were picked from agar plates and inoculated into the wells. A sterilized tooth pick
was used to pic a single colony
11. After inoculation LT plates were covered with a plastic film to prevent evaporation and
incubated with shaking at 150 to 250 rpm overnight at 37˚C
12. After incubation if colour appears then it was LT positive
13. Plates are then washed 2 times with PBS
ST plates:
1. ST plates were also coated with GM1 Elisa and kept overnight
2. GM1 coated plates were washed 2 times with PBS buffer

3. Two hundred µl of 0.1% BSA-PBS added to the plates to block the wells

4. After blocking the plates were incubated at 37˚C for 30 minutes

5. After incubation plated were washed once with PBS.

6. Fifty µl of ST-CB conjugate added to 20 ml of BSA-PBS

42
 7. One hundred µl of ST-CB conjugate + BSA added to each well and incubated at room
temperature for 30 minutes

8. After incubation plates were washed 2 times with PBS

9. Then, 50µl volume of overnight cultures from LT plates were transferred to the wells of
the ST plates.

10. Twenty five µl of anti ST mAb (ST Map) was added to 6 ml of BSA-PBS and then 50µl
of this mixture was added to each well of ST plates. So the total volume in each well is
100µl

11. The plates were then incubated at room temperature for 1:30 hour

Both plates:

1. LT plates were washed 3 times with BSA-PBS tween and 1 time with BSA-PBS

2. Twenty five µl of anti LT mAb (LT map) mixed with 6 ml of BSA-PBS and 50 µl of this
mixture was added to the wells of LT plates.

3. The plates were kept at room temperature for 1:30 hour

4. After 1:30 hour both LT plates and ST plates were washed 3 times with BSA-PBS tween.

5. Thirty seven µl of anti Ig enzyme conjugate is added to BSA-PBS tween which acts as
diluent. 100 µl of this conjugate was added to each well of both LT and ST plates

6. Both plates were washed 3 times with PBS-tween an 1 time with PBS

7. In the meantime OPD substrate solution was prepared by adding 0.015g OPD to 15ml of
Na citrate. Then it was vortexed and 4µl of H2O2 is added to it

8. One hundred µl of OPD mixture was added to every well of both LT and ST plates and
waited for 10 minutes

9. The absorbance was measured at 450nm after 20minutes for LT plates and 10-15 minutes
for the ST plates in a microplate reader

43
Observation:

Figure: LT and ST plates

Results:

If colour appears in LT plates then it is considered as LT positive plates. On the other hand if
ST plates remain colourless then it indicates that plates are ST positive.

In LT plate colour appeared only in 3rd and 4th columns. But 1st two wells of this column were
filled with only positive control. Last 2 wells of 2 nd and 3rd column were filled with negative
control where no colour came. Bacterial colonies were added only in the 2 nd column but no
colour developed in this column. So it indicates the LT toxin was not present in that sample.

In ST plate color developed in 2nd column where the bacterial colonies were inoculated. Color
also developed in the negative controls but no color developed in the positive controls. So it
indicates that result is negative.

Identification of colonizing factors using Immuno Dot blot

technique:
44
A dot blot (or slot blot) is a technique in molecular biology used to detect biomolecules, and for
detecting, analyzing, and identifying proteins. It represents a simplification of the northern
blot, Southern blot, or blot methods.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute watery
diarrhoea in developing countries, particularly among local children less than five years and is
also the most common cause of diarrhea in travelers to ETEC endemic areas. Apart from heat-
labile and heat-stable enterotoxins, ETEC pathogenesis is mediated by specific antigenically
distinct colonization factor antigens (CFAs). Colonization factors (CFs) on the bacterial surface
mediate adhesion to the intestinal epithelium. Several CFAs have been identified; among these,
CFA/I, CFA/II, and CFA/IV are the most common. CFA/II is composed of E.coli surface (CS)
antigens CS1, CS2 and CS3, whereas CFA/IV is composed of CS4, CS5, and CS6. In addition,
several other CFAs have been identified, namely, CS7, CS12, CS14, CS17, and CS21.

Dot blotting technique is done to detect whether the ETEC positive strains are forming
colonizing factors are not the sample of E.coli is first grown on MaConkey agar for
identification. Then PCR is done to detect whether it is ETEC or not. 6 colonies of ETEC
positive sample that have gone through PCR are taken and streaked on the CFA-bile media and
TSA plates(CS21). Then these colonies are suspended in PBS buffer because bacteria don’t show
direct activity in nitrocellulose membrane so it has to be diluted. Then plates are coated with
sample contain the ETEC antigen. Then blocking is done and after that monoclonal antibody,
conjugate are added. Finally color develops by adding the substrate. Each antigen of ETEC
doesn’t have colonizing factors. Those who have colonizing factors will develop color in this
test. E.colihas more than 50 colonizing factors. Here at ICDDR, B, there are antibodies against
13 of them: CS1, CS2, CS3, CS5, CS6, CS7, CS12, CS14, CS17, CS21, CFA1, CFA2 and
CFA3. These are the positive controls. The different colonizing factors have different genes.
Instead of the dot blotting, PCR can also be done. But this is time consuming, expensive and the
bands may overlap.

Procedure:

1. The nitrocellulose membrane sheet was first cut into several strips

45
2. Then the nitrocellulose membranes soaked in PBS buffer and allowed to dry for 5-30
minutes on a blotting paper
3. The strips were then transferred to the wells of dot blot plate
4. Then the positive controls with PBS 4uL are placed on the 2 grids of every strip.
5. Six colonies taken from ETEC positive sample that went through PCR and these colonies
are streaked in CF bile media. Then these were suspended in PBS buffer because bacteria
don’t show direct activity in nitrocellulose membrane so it has to be diluted.
6. Two µl of ETEC positive sample 1 from TSA plates were added to the grids of every
strip in the same place. 2µl of ETEC positive samples from CFA plates are also added to
the grids of every strip. In a total 10 samples were added to every strip and 13
colonization factors of ETEC had to be detected against these 10 samples
7. The strips are then allowed to dry
8. Then the nitrocellulose membrane was blocked by applying 1ml of 1% BSA-PBS.
BSA-PBS will bind to the place in the membrane where sample is not given so that
background activity doesn’t occur when monoclonal antibody is added to the strip.
9. Then it kept in shaker at room temperature for 30-40 minutes
10. BSA-PBS buffer was discarded
11. One thousand µl of 1% BSA-PBS tween was added to each strip in order to be diluted
12. Twenty two µl of monoclonal antibodies specific for each colonizing factor are added to
each strip. Monoclonal antibodies shouldn’t be mixed with each other to prevent
background activity otherwise false positive result may occur
13. Then the plates containing the strips were incubated for 2 hours. During this time
monoclonal antibody which is primary antibody will bind to the sample containing the
antigens
14. The plates were washed 3 times with PBS tween buffer and 1 time with PBS buffer
15. Rabbit anti mouse IgG HRP conjugate which is secondary antibody is diluted into 1:5000
dilutions by adding 26µl of conjugate into 26ml of 0.1% BSA-PBS tween buffer. Then it
is vortexed
16. Nine hundred and fifty µl of conjugate was added to each strip

46
 17. Then plates were incubated for 2 hours in shaker incubator. During this time conjugate
which is secondary antibody will bind to primary antibody
18. After incubation the plates were washed 3 times with PBS tween and 1 time with PBS
buffer.
19. One thousand and seven hundred µl of 4-chlro-1-napthol is diluted in per 10 ml of TBS
20. Five µl of H2O2 was added to this mixture and it is vortexed and then it is poured on the
petri dish
21. Seven µl of mixture which was used as substrate is added to each strip
Enzyme substrate (for 10μl):
 4-chloro-1-napthol, 3mg/ml in 99.9% methanol : 1.7ml

 TBS : 8.3ml

 H2O2 (30%) : 5μl

22. Color development is then observed. The color might turn to light purple and then dark
purple.
23. The plates containing the membrane are washed with tap water for the reaction to stop
The nitrocellulose membrane strips are dried on blotting paper an then stored

47
Observation:

Fi
gure: Immune dot blot assay (color dots are visible on nitrocellulose paper
and data are documented for analysis)

Results:

The color dots were visible on the paper. The more color it gives means that the result is positive
(first 2 dots contain positive +ve control). The possible data are documented for analysis. Every
antigen doesn’t have colonizing factors. Those who have colonizing factors will show positive
color in these strips.

48
Antibiogram to detect sensitivity of different antibiotics:
Purpose: Disk diffusion is a method which is used to detect which organisms are sensitive to
which antibiotics.

An antibiogram is the result of an antibiotic sensitivity test. It is by definition an in

vitro sensitivity, but the correlation of in vitro to in vivo sensitivity is often high enough for the
test to be clinically useful.

Antibiotic susceptibility is the susceptibility of bacteria to antibiotics. Because susceptibility can

vary even within a species (with some strains being more resistant than others), antibiotic
susceptibility testing (AST) is usually carried out to determine which antibiotic will be most
successful in treating a bacterial infection in vivo. Testing for antibiotic sensitivity is often done
by the Kirby-Bauer method. Small wafers containing antibiotics are placed onto a plate upon
which bacteria are growing. If the bacteria are sensitive to the antibiotic, a clear ring, or zone of
inhibition, is seen around the wafer indicating poor growth. Other methods to test antimicrobial
susceptibility include the Stokes method, Etest (also based on antibiotic diffusion), Agar and
Broth dilution methods for minimum inhibitory concentration (MIC) determination. The results
of the test are reported on the antibiogram.

Procedure:

1. Pure Colonies of ETEC was taken from MacConkey agar plate and pure colonies of
V.cholerae are taken from TTGA agar plate by loop

2. Colonies of ETEC was inoculated into 2ml of Muller Hinton(MH) broth and colonies
of V.cholerae are inoculated another 2ml of MH broth

3. Then vortexed to mix properly

4. The broths were incubated at 37˚C for 1-2 hours so that bacteria can grow inside of
the broth

49
 5. MH plates were labelled. Usually 3 plates are used for this test. 2 for ETEC and 1 for
V.cholerae.

6. After incubation sterile cotton swabs were dipped into both broths

7. Then the MH plates are swabbed so that bacteria is spread evenly to the plates. Plates
are first dried in the dryer then they are swabbed with the sample. ETEC from MH
broth 1 are swabbed in MH plate 1 and 2 and V.cholerae from MH broth 2 is swabbed
on MH plate 3.

8. Then antibiotic discs were placed on the MH plates. 6 antibiotic discs were placed on
MH plate 1 against ETEC and other 6 antibiotics are placed on MH plate 2 against
ETEC. 5 antibiotics were placed against V.cholerae in the 3rd MH plate.

9. Plates were incubated at 37˚C for 24 hours.

10. After incubation zone of inhibition was measured in decimetre (dm).

Observation:

Figure: Zone of inhibition was seen around the antibiotic discs

In plate 1 zone of inhibition was observed in following antibiotics:

Cephraxon (cro)- 24

50
Doxaciline (do)-19

Ciprofloxacilin (CIP)- 23

Azithromycin (Azm)- 24

Ampicillin (amp)- 19

Erythromycin (E)- 10

Plate 2:

In plate 2 zone of inhibition was observed in following antibiotics:

Optimazole (SXT)- 26

Tetracycline (T)- 23

Norfloxacilin (nor)- 22

MEL-22

NA- no zone of inhibition appeared

Streptomycin (S)- 13

Plate 3:

Azithromycin (Azm)- 20

Tetracycline (T)- 13

Erythromycin (E)- 17

Ciprofloxacilin (CIP)- 20

Optimazole (SXT)- 7

Results interpretation:

In case of ampicillin from 17dm of zone of inhibition it is able to kill bacteria. In plate 1
the zone of inhibition appeared 19 so it indicates that it is sensitive to ETEC and able to
kill ETEC. If less than 17 appear then it indicates its resistance to ETEC. In case of
Azythromycion its zone of inhibition starts 21 but in plate 3 it appeared 20 so it indicates

51
 that it is intermediate to V.cholerae which means that it can kill V.cholerae but less
effectively. In this way the result is interpreted for other antibiotic. Every has a specific
zone of inhibition from which it is sensitive to bacteria and if it appears less than that
specific point in agar plates then it indicates that it is resistant to bacteria

Salmonella shigelladetection by biochemical tests:

Procedure:
1. Colonies of Shigella taken from SS agar plate and stabbed into MIU media
2. Then Shigella colonies taken from SS agar plate is stabbed and steaked into KIA media
3. Another Shigella colony from SS agar plate is stabbed and streaked into citrate agar
4. Salmonella colony taken from MacConkey agar plate and stabbed into MIU media
5. Then Salmonella colony taken from MacConkey agar plate and stabbed and streaked into
KIA media
6. Another Salmonella colony taken from MacConkey agar plate is stabbed and streaked
into citrate agar
7. Indole paper soaked into paraminobenzyldehyde is added to every MIU media.
8. Then the media are incubated at 37˚C for 24 hours

Observation:

52
Salmonella in MIU Shigella in MIU

Salmonella in KIA Shigella in KIA

53
 S
a l
m o
n ell
a in

citrate agar Shigella in citrate agar

Results:

MIU medium:
MIU medium is used for checking motility, urease production and indole production In case of
salmonella type B, Motility was observed in the media because it was diffused so salmonella is
motile Red color was developed on the top of the media so salmonella is urease positive Inner
portion of filter paper was yellow so salmonella is indole negative
In case of shigellaflexneri,

Motility was not observed in the media because it was stiff so shigella is non-motile Red color
was not developed on the top of the media so shigella is urease negative Inner portion of filter
paper was yellow so shigella is also indole negative

KIA medium:
In case of salmonella type B,

54
The slant was red (alkaline condition). It indicates that salmonella was not able to ferment
lactose so acid was not produced and the slant remained red. This means salmonella is non-
lactose fermenter. Butt was first yellow then it developed black color. It indicates that
salmonella is H2S positive
In case of shigella,
The slant was red (alkaline condition). It indicates thatshigella was not able to ferment lactose so
acid was not produced and the slant remained red. This means shigella is non-lactose fermenter.
Butt was yellow and not changed to black. It indicates that shigella is H2S negative
Citrate agar:
In case of salmonella type B,
The color of the media turned to blue from green which indicates that salmonella is citrate
positive
In case of shigella,

The color of the media remain unchanged which indicates that shigella is citrate negative

Organism Indole Motility Urease Citrate Slant Butt H2S Gas

(MIU) (MIU) (MIU) (KIA) (KIA) (KIA) (KIA)
Salmonell -ve +ve +ve +ve Red slant Yellow +ve +ve
a Spp. (indole (diffuse) (Pink (green (Alkaline (Acidic) Black Crack /
paper color agar ) butt bubble
red top) turns
outside) into
blue)
Shigella -ve -ve -ve -ve Red slant Yellow -ve -ve
Spp. (indole (Stiff) (Agar (Alkaline (Acidic) Yellow No
paper remain ) butt crack
red green) or
outside) bubble

Kligler iron agar (KIA):

55
Kligler Iron Agar (KIA) combines features of Kligler's Lead Acetate medium and Russell's
Double Sugar Agar. It is differential media used to differentiate organisms on the basis of
dextrose and lactose fermentation and hydrogen sulfide production.Phenol red is added as the
color indicator. The basal medium of KIA is composed of casein and meat peptones with the
addition of lactose and dextrose. The production of acid by lactose and/or dextrose fermentation
results in color changes of the phenol red pH indicator.
Lactose fermenting organisms produce yellow slants and butts. There is no reversion to red in the
slant because enough acid is produced to maintain an acid pH under aerobic conditions. Non
lactose fermenters produce red slants and butts. Non-lactose fermenters initially produce a
yellow slant and butt as a result of dextrose fermentation. The concentration of dextrose is only
one percent and, therefore, is rapidly exhausted. Once the dextrose is depleted, the reaction
reverts to alkaline (red slant) due to the oxidation of acids. H2S production results in a blackening
of the medium, either throughout the butt or in a ring formation near the top of the butt. Gas
production is demonstrated by the presence of bubbles or cracks in the medium.

Composition:

Ingredients per liter of deionized water:

Peptone 15.0gm
Lactose 10.0gm
Proteose Peptone 5.0gm
Sodium Chloride 5.0gm
Beef Extract 3.0gm
Yeast Extract 3.0gm
Dextrose 1.0gm
Sodium Thiosulfate 0.3gm
Ferrous Sulfate 0.2gm
Phenol Red 0.024gm
Agar 12.0gm

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Final pH 7.4 +/- 0.2 at 25ºC.

Motility Indole Urea (MIU):

MIU Medium Base is used to detect motility, urease and indole production in single tube. Casein
enzymichydrolysate provide amino acids and other nitrogenous substances. Sodium chloride
maintains osmotic equilibrium. Dextrose is fermentable carbohydrate. Phenol red is the pH
indicator which turns pink- red in alkaline conditions. The test cultures are stab-inoculated.
Motility and urease reactions are read before testing Indole production. Motile organisms show
either diffused growth or turbidity extending away from stab inoculation line while nonmotile
organisms grow along the stabline. Organisms that utilize urea produce ammonia which makes
the medium alkaline, showing pink-red colour by change in the phenol red indicator. Indole is
produced from tryptophan present in casein enzymichydrolysate. The indole produced combines
with the aldehyde present in the Kovac's reagent to form a red complex.

Composition:

Ingredients Gms / Litre

Casein enzymichydrolysate- 10.000

Dextrose- 1.000

Sodium chloride- 5.000

Phenol red -0.010

Agar- 2.000

Final pH (at 25°C) 6.8±0.2

Simmons citrate agar:

57
Simmons Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy.
Ammonium Dihydrogen Phosphate is the sole source of nitrogen. Dipotassium
Phosphate acts as a buffer. Sodium Chloride maintains the osmotic balance of the
medium. Sodium Citrate is the sole source of carbon in this medium. Magnesium Sulfate is a
cofactor for a variety of metabolicreactions. Agar is the solidifying agent. Organisms capable of
utilizing ammonium dihydrogen phosphate and citrate will grow unrestricted on this medium. If
citrate can be used, the microbe will accumulate alkaline/basic byproducts.

Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of

converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for
the production of energy. Growth is indicative of utilization of citrate, an intermediate
metabolite in the Krebs cycle.

When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia,

which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium
from green to blue above pH 7.6.

Composition:
Ingredients per liter of deionized water:*

Sodium Chloride (NaCl) 5.0 gm

Sodium Citrate (dehydrate) 2.0 gm

Ammonium Dihydrogen Phosphate 1.0 gm

Dipotassium Phosphate 1.0 gm

Magnesium Sulfate (heptahydrate) 0.2 gm

Bromothymol Blue 0.08 gm

58
 Agar 15.0 gm

Deionized water-100ml, Final pH 6.9 +/- 0.2 at 25 degrees C.

Abbreviation
 ICDDR, B- International Centre For Diarrheal Disease Research, Bangladesh
 ELISA- Enzyme Linked Immunosorbant Assay
 GM1- MonosialosylGanglioside
 HRP- Horse-Radish Peroxidase
 Ig- Immunoglobulin
 LPS- Lipopolysaccharide
 CT- Cholera toxin
 OD- Optical Density
 PBMC- Peripheral Blood Mononuclear Cell
 PBS- Phosphate-Buffered Saline
 RBC- Red Blood Cell
 Rpm- Rotation Per Minute
 ALS- Antibody in Lymphocyte Supernatant
 APC- Antigen Presenting Cells
 BSA- Bovine Serum Albumin

59
Conclusion:

From the work experience, I have learned about many aspects of immunological and molecular
techniques and use of equipment as they were provided exposure into the internal laboratories
and briefed with necessary information about the system. It was simply a great experience to me.
I have grown much during these10 days of journey under these institutions. My theoretical
knowledge has been put to practice. Furthermore, I got the opportunities to meet with many
people who are extremely talented and motivated.

60
Reference

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Health Organization; 1992.Publication no.WHO/CDD/SER/80.4 Rev 4.
2. Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of
Vibrio cholera. Atlanta, Georgia: CDC, 1994.
3. World Health Organization. Manual for the laboratory investigations of acute enteric
infections. Geneva: World Health Organization, 1987; publication no. WHO/CDD/83.3
4. McLaughlin JC. Vibrio. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yolken
RH, ed. Manual of clinical microbiology. Washington, DC: ASM Press; 1995:465-476.
5. Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of
Vibrio cholera. Atlanta, Georgia: CDC; 1994.
6.Guidelines for Biosafety in Teaching Laboratories (American Society for Microbiology 2012).
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WHO / 2003

7. Guidance on regulations for the Transport Infectious substances 2011-2012

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