Internship reportSubmitted by: MD. MINHAZUR RAHMAN

Course Name: Industrial Attachment (BTE400)

Internship report on

I

Submitted by MD.MINHAZUR RAHMAN

ID; 11336002Department of Mathematics and Natural Science

Submitted to

Department of Mathematics and Natural Sciences BRAC University

Summary

As a part of my graduation I required an industrial attachment and with the help BRAC

University I started my internship at Institute for Developing Science and Health Initiatives

(ideSHi) and immunology laboratory of The International Centre for Diarrheal Disease Research

for 2 weeks. In each organization I had to pass 7 days.

This internship program gives me the chance to mixed the knowledge with the working sector

and with different people in different environment. In this program each day I was introduced

with the different laboratory works like PCR, ELISPORT, ELISA etc.

After completing this internship each student will have to submit a training report duly endorsed

by the authority of the respective enterprise. this knowledge and experience provided a broad

view of internal laboratories and briefed with necessary information about the system. In

addition, the students are benefited in terms of the technical details provided by the companies.

Content

Introduction…………………………………………………………………………………………………….. 1 Organizations………………………………………………………………………………………………….. 2 Report on ideSHi laboratory 3 TP Test……………………………………………………………………………………………………………… 4 Isolation of peripheral blood mononuclear cells PBMCs…………………………………… 5 Ficoll Gradient Centrifugation…………………………………………………………………………… 5 RBC Lysis Method…………………………………………………………………………………………….. 5 PCR………………………………………………………………………………………………………………….. 7 Genomic DNA extraction from human blood sample……………………………………….. 7 Gel Electrophoresis………………………………………………………………………………………….. 8 Isolation protocol (AREI)………………………………………………………………………………….. 9 Different types of media used in diagnostic microbiology……………………………….. 10 Acute Respiratory Infection Protocol……………………………………………………………….. 12 Diarrhea Protocol…………………………………………………………………………………………….. 13 Enteric Fever Protocol……………………………………………………………………………………… 14 Gram staining…………………………………………………………………………………………………… 15 Report on icddr,b Immunology Labor 16 Enteric disease analysis in Immunology Laboratory………………………………………….. 17 Specimen Culture and Isolation………………………………………………………………………… 18 Media for specimen culture……………………………………………………………………………… 19 Flow chart for identification of Vibrio cholerae and ETEC…………………………………. 20 Serotyping of V. Cholerae…………………………………………………………………………………. 21 Dark field microscopy……………………………………………………………………………………….. 22 PCR for ETEC…………………………………………………………………………………………………….. 23 Gel Electrophoresis for ETEC…………………………………………………………………………….. 24 GM1 ELISA………………………………………………………………………………………………………… 25 LPS and CT ELISPOT Assay………………………………………………………………………………… 29 Quick Dot Blot………………………………………………………………………………………………….. 31 Flow Cytometry………………………………………………………………………………………………… 32 Conclusion………………………………………………………………………………………………………… 37 References……………………………………………………………………………………………………….. 37

IntroductionThis report is a short description of an internship program (duration 2 weeks), which is a part of

the course called Industrial Attachment, course ID: BTE 400. This is required for completing

B.Sc. Biotechnology at BRAC University. The internship was carried out at icddr.b and ideSHi.

The range of the work carried out at the laboratory was varied and divided among multiple

scientists. Therefore, there was no specific area of focus and rather the terms of the internship

required me to spend time with the scientists in charge of their respective projects for a specific

time period.

Learning goals from this internship:

Learning the working conditions of an industrial research lab.

Gather the experience of working in a professional environment.

Using the skills and knowledge that I have gathered during the course of my education as

an undergrad Identifying my shortcomings in comprehension and skill within a

professional environment and learning how to overcome them.

Learning research methodologies (field work observation, collection and assay of

relevant data etc.) and the organization of a research project.

Accumulating experience in working with people from different disciplines.

This report also carried the short description of those organizations.

Organization:01. The International Centre for Diarrheal Disease Research (icddr,b), Immunology laboratory.

02. Institute for Developing Science and Health Initiatives (ideSHi)

The International Centre for Diarrheal Disease Research

(icddr,b), Immunology laboratory.The aims of icddr,b is to harness the power of high-quality research to address the health

problems of Bangladesh and the developing world more generally. For more than 50 years,

icddr,b has been a beacon of high-quality research in South Asia. It has led research that has

saved millions of lives. 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.

Ithe work of this organization is guided by a number of key principles-

High-quality science: icddr,b is committed to scientific excellence, and disseminating

information through the scientific literature including high-impact journals.

Local relevance: The questions icddr,b addresses are important priorities for Bangladesh and

regionally.

Policy-oriented research: To achieve practical impact, our research has a strong focus on the

realities of implementation in resource-poor settings.

Global networking: icddr,b has outstanding links to world-leading research centers in the

developed world, enabling us to access unmatched global knowledge and expertise.

Regional and global impact: As well as enhancing public health and clinical care in

Bangladesh, we also aim to work with other centers in the developing world to build knowledge

and promote the uptake of evidence-based policy and practice.

Training and capacity-building: We make an important contribution to the regional science

base, training of local healthcare professionals, and to research and public health capacity in the

developing world more generally

Institute for Developing Science and Health Initiatives

(ideSHi)

Institute for Developing Science and Health Initiatives (ideSHi)was established in 2012 by Dr.

Firdausi Qadri is the founder of this institute and also the winner of Christophe Mérieux Prize.

The goal of ideSHi is to strengthen scientific capacity and encourage innovations to make

Bangladesh a local leader in the field of health and multi-disciplinary research globally. ideSHi

focuses on development of local scientists and scientific capacity, scientific research, public

health initiatives and educational programs.

The mission of this institute is towards improved lives and better health for people in Bangladesh

and other developing countries.

In the immunology of the icddr,b task and the research are divided into several segments like,

01.Speciman culture and isolation of the organisms.

02. serotyping of the V.Cholera.

03. Dark field microscopic test.

04. Gel electrophoresis.

05. GM1 ELISA

06. ELISPORT Assay

07. LPS and CT

08. Quick Dot Blot.

09. Flow Cytometry.

Specimen Culture and IsolationVibrio cholerae has more than 200 serogroups. For its serotyping, antiserum is used. At icddr,b 3

serogroups are identified. They are: O139, O1 Ogawa and O1 Inaba. Enterotoxigenic Escherichia

coli (ETEC) is the leading bacterial cause of diarrhea. ETEC is responsible for producing toxins

like LT and ST.

Different agar plates are used as media to isolate the V. cholerae and E.coli from the sample of

the patients.

MacConkey Agar: MacConkey agar is a selective and differential culture medium for

bacteria designed to selectively isolate Gram-negative and enteric bacilli and differentiate them

based on lactose fermentation. The crystal violet and bile salts inhibit the growth of gram-

positive organisms which allows for the selection and isolation of gram-negative bacteria.

Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate

lactose, and the pH indicator neutral red. MacConkey agar is one of the most common agar

plates found in a clinical microbiology lab.

Taurocholate Tellurite Gelatin Agar (TTGA): The TTGA is a selective and

differential agar that is used to isolate Vibrio cholerae. Overnight growth of V. cholerae on

TTGA agar, small cloudy 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 biochemical are needed to isolate

particular species from this medium.

Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar: Thiosulfate-citrate-bile salts-

sucrose agar or TCBS agar is a type of selective agar culture plate that is used in microbiology

laboratories to isolate Vibrio spp. TCBS Agar is highly selective for the isolation of V. cholerae

and V. parahaemolyticus as well as other vibrios. TCBS agar contains high concentrations of

Sodium thiosulfate and Sodium citrate to inhibit the growth of Enterobacteriaceae. Inhibition of

Gram-positive bacteria is achieved by the incorporation of ox gall, which is a naturally occurring

substance containing a mixture of bile salts, and sodium cholate, a pure bile salt. Sodium

thiosulfate also serves as a sulfur source and, in combination with ferric citrate, detects hydrogen

sulfide production. sucrose is included as a fermentable carbohydrate for the metabolism of

vibrio’s. The alkaline pH of the medium enhances the recovery of V. cholerae and inhibits the

growth of others. Thymol blue and bromothymol blue are included as indicators of pH changes.

Flow chart for the identification of Enterotoxigenic E.Coli

Flow chart for the identification of V. Cholerae

Sample from the patient

Culture on MacConkey plate

E.Coli Specific colony was tested for ETEC

Detection of ST or LT by PCR

Detection of toxin phenotype by GM1 ELISA

Detection of CFs by Dot Blot

Dark Field MicroscopyBiological dark-field microscope was used to identified the bacteria in primary level. In a dark

field microscope, a sample is placed on the stage it appears bright against a dark background.

This is a method which excludes the unscattered beam from the image. As a result, the field

around the specimen is generally dark.

Procedure: 1. Small drop of test stool sample was placed on a slide and cover slip was applied.

2. A specimen in dark field, an opaque disc was placed underneath the condenser lens, so that

only light that is scattered by objects on the slide can reach the eye.

3. presence of the bacteria can be seen.

Dark field microscopy

Culture on the TTGA or TCBS plate

Serogroup identification of V.Cholerae by serology

test

Figure: Presence of bacteria under Dark Field Microscopy

Serotyping of V. Cholerae

Monoclonal antibodies technique is helpful for the serotyping of the V. Cholerae. For serotyping

of V. Cholerae O1- Ogawa, V. Cholerae O1- Inaba, or V. Cholerae O139 specific monoclonal

antibodies (MAb) were used.

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

2. Ogawa, V. cholerae O1- Inaba, and V. Cholerae O139 specific MAb was placed on the slide.

3. With a loop of half of the colony from the TTGA plate was applied on the slide where

antibody is present.

4. Agglutination was observed when specific colony for the antisera is present.

5. The occurrence of clear agglutination within 2 minutes was considered as positive result.

PCR for ETEC

PCR for the identification of Entero toxigenic E. coli

Multiplex PCR was used for the identification of Entero toxigenic E. coli. ‘Multiple Template

PCR Reaction’ was executed in this process for HTS, HTP and LT pathogenic genes. MPCR

was performed to amplify the desired aforementioned genes of ETEC.

Template Preparation 1. Six colonies of ETEC from MacConkey agar was taken by needle/toothpick.

2. Colonies were kept in 100µl PBS solution.

3. Vortex was done to mix the organisms with PBS.

4. Tubes were boiled at 100°C for 10 minutes in a water bath to cell lysis.

5. The tubes were kept in ice for 1 minutes after the heating.

6. Centrifugation was done for 10 minutes at 12000rpm (20°C) 7. The 1.5µl supernatant was

transferred as template DNA.

PCR Master Mix

Template 1.5µl

Deionized d.H2O 10.85µl

PCR buffer with MgCl2 (10x) 2.5µl

dNTPs (2.5mM each) 4.0µl

Primers: LT, STh, Stp mixture (4pm/ µl) 2.0x3= 6µl

MgCl2 (25mM) 0.5µl

Taq polymerase (5U/µl) 0.15µl

Total 24.00 µl+ 1.5µl

Gel Electrophoresis

PCR product that was ran on the agarose gel to observe and compare the bands of DNA.

Typically, gels made from polyacrylamide are used to separate proteins on the basis their

different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in

order to unravel the protein. SDS is a detergent that gives all the proteins the same overall

negative charge so that when an electric current is applied to the gel, separation is only due to the

size of the protein. The result of the electrophoresis was used to compare the obtained bands with

the ladder and accordingly the identification was completed. It was confirmed that the E. coli

contains the same toxin genes.

Agarose Gel preparation:01. Added the needed amount of powdered agarose (typically 8-15%) to the electrophoresis

buffer (TBE or TAE) in an Erlenmeyer flask. The buffer should not occupy more than 50% of

the volume of the flask.

02. Heat the agarose solution in a microwave oven or in water bath to allow all of the grains of

agarose to dissolve. If part of the buffer evaporated during the heating, bring the solution back to

the original volume through the addition of buffer.

03. Cool down the solution to 60 C.

Figure: Different band of DNA

Procedure: 01. Agarose Gel Electrophoresis performed.

02. 3µl loading dye was mixed with 4.5µl PCR product.

03. Amplified PCR products were separated at150 volt for 30 minutes.

04. GelRed was used to stain the gel for gel-view and the bands were observed on a Gel

documentation system (BIORAD) under UV.

Size of toxin products

LT 300bp

STp 166bp

STh 100bp

GM1 ELISA

ST/LT Testing of ETEC Isolated in Clinical Specimens Detection of E. coli heat-stable (ST) and heat-labile toxins produced by clinical isolates of

enterotoxigenic E. coli (ETEC).

E. coli LT+ strains are identified using a direct GM1 ELISA. Briefly, bacteria are cultivated

overnight in medium containing ELISA wells, previously coated with ganglioside GM1.

Aliquots from each well is transferred to STCTB-GM1 conjugate-coated wells of a separate

ELISA plate and used for the analysis of ST. The plate used for overnight culture is subsequently

incubated with monoclonal antibody directed against LT, enzyme labeled goat anti-mouse

immunoglobulin, and substrate. An absorbance of 0.1 above background is considered as a

positive result.

E. coli ST+ strains are identified using inhibition ELISA. Briefly, a monoclonal antibody

directed against ST is added to the STCTB-GM1 conjugate-coated wells containing overnight

cultures of E. coli. The wells are subsequently incubated with enzyme labeled goat anti-mouse

immunoglobulin and substrate. Inhibition of ≥50% of the absorbance value, as compared to the

mean absorbance value obtained with two ST-negative reference strains is considered as positive

result.

Figure: ELISA

Procedure 1. E. coli isolates were cultured on MacConkey agar plates. Differentiated streaks were used to

identify at least 6 individual colonies. The plates were incubated at 37°C overnight.

2. 100µl of ganglioside GM1 was added (0.5µg/ml to each well of an ELISA plate) and was

incubated at 37°C for 4 hours. GM1-coated wells were washed twice with PBS. The wells were

blocked by the addition of 200µl of 0.1% BSA-PBS at 37°C for 30 minutes.

3. Washing once with PBS was done and 100µl of supplemented LB broth was added.

4. 4 reference strains were inoculated, 2 single colonies for each strain. Then the inoculation of 6

individual colonies was done from each agar plate. A wooden stick was used to inoculate one

bacterial colony/well in the ELISA plate. The column 1 and 12 and rows A and H in the ELISA

plate were excluded.

5. The plate was covered with a plastic film and was incubated with shaking at 250 rpm

overnight at 37°C (LT plates)

6. The turbidity of the wells were registered, wells those were lack of visible growth were

excluded.

7. A new GM1 coated plate was washed (for each LT plate) twice with PBS. Blocking wa done

with 200µl 0.1% BSA-PBS at 37°C for 30 minutes (ST plates).

8. The ST plates were washed once with PBS. 100µl recombinant ST- CTB conjugates solution

was added. Incubation was done at room temperature for 60 minutes.

9. The ST plates were washed 3 times with PBS. 50µl volume of the overnight cultures from the

LT plates were transferred from the LT plates to the corresponding wells in the STplates, 50µl of

anti-ST MAb ST 1:3 solution was immediately added. Shaking was done and plates were

incubated at room temperature for 90 minutes.

10. The LT plates were washed 3 times with PBS-0.5% Tween. 100µl of anti-LT MAb LT

39:13:3 was added and incubated at room temperature for 90 minutes.

Result AnalysisLT plates

• The background is defined as the mean absorbance determined for LT-negative control strains.

• A positive result is an A450 nm value of ≥mean background + 0.1, indicate on each original

protocol sheet the cut off level for positive samples.

ST plates

• The background is defined as the mean absorbance determined for LT-negative control strains.

• The calculation is done by 50% inhibition concentration value (IC50).

Test Results At least 2 out of 3 colonies must shows visible growth, wells with no growth will be excluded.

1. ST positive result: ≥50% inhibition of the absorbance value measured as compared to the

absorbance value obtained with negative reference strains.

2. LT positive result: absorbance value of ≥ 0.1 above background.

LPS and CT ELISPOT Assay

The frequency of immunoglobulin-secreting B cells can be determined with the ELISpot assay.

B cells secreting antigen-specific immunoglobulin, or indeed immunoglobulin of all specificities,

can be quantified using this assay.

Figure: CT ELISPOT Assay

Procedure Coating

01. Nitrocellulose plates were coated with 100µl of: ,

GM1 in PBS for Cholera toxin

LPS PBS

Goat anti human IgGX PBS for the total- immunoglobulin plate for

positive control

KLH in PBS for negative control

02. Plates were incubated at 4°C overnight

03. The GM1 coating buffer was decanted and the CT plate was washed 3 times with PBS.

Excessive liquid was removed. 100µl of recombinant cholera toxin B-subunit (rCTB) was added

to each of the GM1 coated wells. The plate was incubated for one hour at 37°C.

Blocking

01. Plates were washed twice gently with PBS only

02. Next the plates were blocked for 2 hours with 200µl RPMI complete media at 37°C

MNC collection

After peripheral blood mononuclear cells (PBMC) were isolated from whole blood by ficoll

gradient. Total and antigen (CTB and LPS)-specific IgG, IgA and IgM ASC were detected

simultaneously on nitrocellulose membrane.

Loading of samples and plate developing

1. The blocking solution was decanted and 50µl of fresh RPMI complete was added in each well.

2. Then 50µl of cell suspension (5x105 cells) was added to all the antigen specific wells and 10µl

and (5x105 cells) was added for total Ig.

3. The plates were incubated with cells for 3 hours at 37°C in a CO2 (5%) incubator.

4. The cells were next decanted and then washed 4 times with PBS-Tween (0.05%) and 2 times

with PBS. Excessive liquid was removed (without making the membrane completely dry)

5. Anti-human IgG and Anti-human IgA-HRP mixture were diluted at 1:500 and anti-human

IgM-HRP was diluted at 1:500 in 1% FBS in PBS-Tween 0.05% and 100µl was added to each

well. The plates were incubated overnight at 4°C.

6. The plates were washed 4 times with 0.05% PBS-Tween and 2 times with PBS.

7. The HRP substrate was prepared by adding H2O2 to 3-Amino-9-Ethyl-carbazole- AEC.

8. 100µl/well of BCIP/NBT substrate was added to every well and the color reaction was

immediately monitored at low magnification stereomicroscope. The color reaction was stopped

once the spots were clearly visible.

9. The color reaction was stopped once the spots were clearly visible. The substrate was decanted

and the plates were washed in tap water repeatedly and shaken off to remove excess water. These

plates were finally soaked in tap water for 30 minutes before letting them dry at room

temperature.

10. The spots were counted under low-magnification and the plates stored away from light for

automated counts later.

11. Each red spot represented an IgA and IgM secreting cell while every blue spot indicated and

IgG secreting cell.

Quick Dot Blot

Detection of Colonization factor (CF) by immuno Dot blot assay Dot blotting is a procedure to observe the colonization factor. In this process colonizing factors

are the receptor with which E. coli attaches to the gut.

Dot blotting is a type of western blot. It is done to detect proteins on the bacterial cell wall. The

enterotoxic E.coli have colonizing factors that help them in forming a colony. E.coli has more

than 50 colonizing factors. Here 13 of them are used CS1, CS2, CS3, CS5, CS6, CS7, CS12,

CS14, CS17, CS21, CFA1, CFA2 and CFA3. These are the positive control. The protein binds to

the nitrocellulose paper.

Procedure 1. The nitrocellulose membrane was soaked in PBS and allowed to dry for 5-30 minutes.

2. 2µl fresh frozen control bacteria (duplicates) diluted in PBS (about 4-10 MC Farland) were

applied. It was dried for 5 minutes.

3. The membrane was blocked in 1% BSA-PBS for 20 minutes at room temperature (1825°C)

with slow rocking.

4. The blocking solution was discarded and antibody were added (monoclonal 1:5- 1:100) diluted

in 0.1% BSA-PBS with 0.05% Tween 20 for 2 hours at room temperature with slow rocking.

5. Washing 3 times for 5 minutes was done with PBS-Tween. The HRP-enzyme conjugate was

added. (Jackson anti-mouse IgG, twice as high concentration as for ELISA) Incubation was done

at room temperature for 2 hours with slow rocking.

6. Washing 3 times for 5 minutes was done with PBS-Tween and once with PBS. It was

developed with 4-chloro-1-napthol –H2O2 for 10 minutes.

Enzyme substrate (for 10µl):

4-chloro-1-napthol, 3mg/ml in 99.9%

methanol

1.7ml

TBS 8.3ml

H2O2 (30%) 5µl

7. The membrane was washed with tap water and then dried.

8. The result was recorded.

Flow Cytometry

Human B and T cell detection and counting by Flow Cytometry

Flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting,

biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing

them by an electronic detection apparatus. In the icddr.b immunology laboratory, phenotyping

by flow cytometry was done for stained and unstained PBMC.

Figure: Flow Cytometry Result After Analysis

Procedure:1. PBMCs (~1x106 lymphocytes) were obtained after ficoll separation, washing and cell

counting of blood specimen.

2. Half of the lymphocytes were transferred into a 96-well V-bottom plate as control.

3. Rest half of the lymphocytes were transferred into another well (stained)

4. Each well was filled up to 200µl with FACS buffer.

5. Centrifugation of V-bottom plate was done at 700g for 5 minutes at 4°C

6. The supernatant was pipetted out and discarded.

7. Abs was added at 1:10 dilution in FACS buffer, final volume was made 20µl in stained well

and 20µl buffer only in the control well.

8. Incubation was done at 4°C for 45 minutes in dark

9. 200µl FACS buffer was added and centrifugation was done at 700g for 5 minutes at 4°C

10. The supernatant was discarded and 400µl cell fix was added to each tube.

11. The FACS machine was used to determine the amount.

Genomic DNA extraction from human blood sample

Blood sample collected in EDTA-containing vacutainer tubes for chemical processing. The white blood cells, mono nuclear cells contains genomic DNA. The red blood cells are eliminated by lysis. Blood is collected immediately or the stored blood is used. The chemical treatment will be used to extract the DNA to store or further use. The standard chemicals are used for a successful procedure.

Procedure 1. Blood sample was collected and kept in a Heparin tube. 2. Liquid blood venogects were agitated gently in a rotating blood mixer / vortex. 3. 500µl blood was poured into a 1.5 ml eppendorf tube and 1 ml of red blood cell lysis buffer was added. 4. The microfuge tubes were homogenized by shaking and centrifugation for 2 minutes at 7000rpm. 5. The supernatant was discarded and the step 3-5 were repeated for two times to remove hemoglobin. The pellet was broken down by vortexing and rinsed in RBC lysis buffer. 6. The tube was placed downward on a paper towel for few seconds to remove the liquids. 7. 400 l of nucleic lysis buffer was added to the eppendorf and the pellet were dissolved by pipetting. 8. 100 l saturated NaCl (5M) and 600 l of chloroform were added to the eppendorf tube and mixed by vortexing. 9. Centrifugation was performed for 2 minutes at 7000rpm. 10. 400 l of supernatant was transferred to a new 1.5ml tube. 11. 800 l of cold (-20°C) absolute ethanol was added and shaking was done. Vortex was performed until a mucus like appearance of the DNA. 12. Centrifugation was performed for 1 minutes at 12,000rpm to precipitation. 13. The supernatant was discarded and the tube was dried in room temperature by keeping downward on a paper towel. 14. 50 l of deionized distilled water / TE was added to the extracted DNA and vortexed to store at -4°C and -20°C.

PCR Multiplex PCR was used for the identification of Entero toxigenic E. coli. ‘Multiple Template

PCR Reaction’ was executed in this process for HTS, HTP and LT pathogenic genes. MPCR

was performed to amplify the desired aforementioned genes of ETEC.

Template Preparation 1. Six colonies of ETEC from MacConkey agar was taken by needle/toothpick.

2. Colonies were kept in 100µl PBS solution.

3. Vortex was done to mix the organisms with PBS.

4. Tubes were boiled at 100°C for 10 minutes in a water bath to cell lysis.

5. The tubes were kept in ice for 1 minutes after the heating.

6. Centrifugation was done for 10 minutes at 12000rpm (20°C) 7. The 1.5µl supernatant was

transferred as template DNA.

PCR Master Mix

Template 1.5µl

Deionized d.H2O 10.85µl

PCR buffer with MgCl2 (10x) 2.5µl

dNTPs (2.5mM each) 4.0µl

Primers: LT, STh, Stp mixture (4pm/ µl) 2.0x3= 6µl

MgCl2 (25mM) 0.5µl

Taq polymerase (5U/µl) 0.15µl

Total 24.00 µl+ 1.5µl

Gel Electrophoresis

PCR product that was ran on the agarose gel to observe and compare the bands of DNA.

Typically, gels made from polyacrylamide are used to separate proteins on the basis their

different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in

order to unravel the protein. SDS is a detergent that gives all the proteins the same overall

negative charge so that when an electric current is applied to the gel, separation is only due to the

size of the protein. The result of the electrophoresis was used to compare the obtained bands with

the ladder and accordingly the identification was completed. It was confirmed that the E. coli

contains the same toxin genes.

Agarose Gel preparation:01. Added the needed amount of powdered agarose (typically 8-15%) to the electrophoresis

buffer (TBE or TAE) in an Erlenmeyer flask. The buffer should not occupy more than 50% of

the volume of the flask.

02. Heat the agarose solution in a microwave oven or in water bath to allow all of the grains of

agarose to dissolve. If part of the buffer evaporated during the heating, bring the solution back to

the original volume through the addition of buffer.

03. Cool down the solution to 60 C.

Procedure: 01. Agarose Gel Electrophoresis performed.

02. 3µl loading dye was mixed with 4.5µl PCR product.

03. Amplified PCR products were separated at150 volt for 30 minutes.

04. GelRed was used to stain the gel for gel-view and the bands were observed on a Gel

documentation system (BIORAD) under UV.

Figure: Different band of DNA

Procedure 1. ELISA plates were coated with MP (membrane preparation) antigen. The antigens are then added to ELISA plate (100µl/well). Plates are then kept at room temperature overnight. 2. After overnight incubation, plates were washed 3 times with PBS.

3. 200µl of 1% BSA-PBS-Tween was added to each well. The plates were then incubated at 37°C for 30 minutes. 4. After 30 minutes, the plates were washed 3 times with PBS-Tween and once with PBS. 5. 0.1% BSA-PBS Tween were added (100µl/well) are added to 3 wells which were the negative control. 6. Pool plasma were added into 3 wells (100µl/well) as the positive control (1:100 dilution using 0.1% BSA-PBS Tween as diluent) 7. 24 hours/ 48 hours ALS (Antibody in Lymphocyte Secretion) sample of patients were added in other wells in 1:2 dilutions. 8. The plates were then incubated at 37°C for 1.30 hours 9. After 1.30 hours incubation the plates were washed 3 times with PBS-Tween and once with PBS. 10. 100µl/well Anti-Human IgA HRP (Horse Radish Peroxidase) were added to each well (1:1000 dilution using 0.1% BSA-PBS Tween as diluent). The plates were then incubated at 37°C for 1.30 hours. 11. After 1.30 hours the plates were washed 3 times with PBS-Tween and once with PBS. 12. 0-Phenylenediamine (OPD) was used as substrate for Horse Radish Peroxidase. In per 10.0ml Na-Citrate solution 0.01 g OPD was added. 13. To 10.0ml of OPD solution 4.0ul H2O2 was added. 14. Then 100µl of substrate solution was added to each well. 15. Then reading of the plates are taken at 450nm in ELISA reader. Isolation of peripheral blood mononuclear cells PBMCs (Ficoll Gradient Centrifugation Method ) 1. The heparinized venous blood (3ml) was diluted with same volume of PBS. 2. The diluted blood was gently added onto the Ficoll (2/3rd volume of the blood). It was 6ml diluted blood and 4ml Ficoll. 3. The centrifugation was performed at 772g for 25 minutes at 20°C. The acceleration was 9 and deceleration was 1. (The centrifugation was balanced by another tube) 4. Four layers were formed: from the bottom – RBC, Ficoll, MNC and plasma. 5. The plasma was transferred into a labelled falcon tube for further use. 6. The MNCs were removed on the top of the Ficoll layer carefully with a pipette and discard the RBCs. 7. The cells were washed once with 10ml PBS and centrifuged at 953g for 10 minutes at 20°C (Acceleration 9 and deceleration 9). 8. Then the cells were re-suspended at in 10ml of PBS. Then mixed well to collect for 2nd wash and 25μl of from here are taken for cell count. 9. The cells were counted in25 large squares of the hemocytometer, by using 25μl cells with 25μl of Trypan blue. 10. For each million cells 100μl RPMI media is required and minimum of 80μl of media was added to culture plate. 11. The Falcon tube is then again centrifuged at force of 953G, acceleration 9, deceleration 9 for 10minutes at 20°C. Supernatant were removed and pallet were collected with RPMI media.

12. The cells were cultured in the culture plate for 48 hours at 37°C with 5% CO2 for antibody secretion for ELISA RBC Lysis Method

1. The blood sample was collected and the volume was measured. 2. Lysis solution (NH4Cl) was added in 1:20 ratio. (For 2.4ml blood- 48ml lysis solution) 3. The tube was kept in room temperature for 5 minutes exactly. 4. Centrifugation was done at 953g for 5 minutes. 5. The supernatant was discarded and ice cold PBS was added until the volume was 10.0ml 6. Centrifugation was done at 953g for 5 minutes. 7. The supernatant was discarded and RPMI complete media was added to cell pellet. 8. The cells were kept in the culture plate at 37°C in 5% CO2 for antibody secretion.

Different types of media used in diagnostic microbiology Tryptic Soy Broth Tryptic Soy Broth (TSB) is used for the cultivation of a wide variety of microorganisms. Tryptic Soy Broth conforms to Harmonized USP/EP/JP Requirements. This medium was originally developed for use without blood in determining the effectiveness of sulphonamides against pneumococci and other organisms. Clostridia and non-sporulating anaerobes grow luxuriantly in this broth when incubated under anaerobic conditions.

Salmonella Shigella Agar Salmonella Shigella Agar or SS agar is used for the isolation of Salmonella spp. and some strains of Shigella spp. This media is a modification of the Desoxycholate Citrate Agar. It is recommended for testing clinical specimens and food testing for the presence of Salmonella spp. and some Shigella spp.comparison study of a rapid antigen test.

Kligler Iron Agar (KIA) It is a differential media used to differentiate microorganisms on the basis of dextrose and lactose fermentation and hydrogen sulphide production. It is also recommended for differentiation of enteric Gram-negative bacilli from clinical specimens and food samples.

Thiosulfate-citrate-bile salts-sucrose agar Thiosulfate-citrate-bile salts-sucrose agar or TCBS Agar is used for the selective isolation of Vibrio cholerae and other enteropathogenic vibrios. TCBS Agar is recommended for isolating Vibrio spp. from stool specimens, and specified in standard methods for food testing.

Todd Hewitt Broth Todd Hewitt Broth is used for the cultivation of streptococci and other fastidious microorganisms. It also identifies of group A streptococci from throat cultures by fluorescent-antibody. The media is recommended as an enrichment medium for growth of streptococcal cells in the identification of groups A and B by Immuno-fluorescence staining and also for group A streptococci in a Blood Agar Blood agar contains general nutrients and 5% sheep blood. It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism. Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins.

Cary Blair Cary-Blair Medium is used to transport rectal swabs and fecal specimens to isolate enteric pathogens.

Mueller Hinton Agar Mueller-Hinton Agar isolates pathogenic Neisseria species. The media is mainly used for Antimicrobial Susceptibility Testing (AST). This media can come in the form of broth as well.

Motility Indole Urease (MIU) Motility Indole Urease is a multi-purpose medium for differentiation of enterobacteriacae that combines three individual tests into a single medium. It is used to detect motility, urease and indole production in a single tube. The test cultures are stab-inoculated. Organisms that are motile show either diffused growth or turbidity extending away from inoculated stab line. Nonmotile organisms grow along the line of stab. Urea utilizing organisms produce ammonia, making the medium alkaline. This shows a pink-red color due to the presence of phenol red indicator. Indole is produced from tryptophan. The indole combines with the aldehyde present in the media and form a red complex.

Gram staining Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolonization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolonization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves these steps:

Procedure

1. Smear of bacterial cells was made on a marked glass slide and was heat fixed. 2. Cells were stained with crystal violet dye and after 1 minute it was washed with water. 3. Next, a Gram's iodine solution (iodine and potassium iodide) was added and washed after 1 minutes with water. 4. A decolorizer, acetone was added to the sample, which dehydrates the peptidoglycan layer and was washed immediately. 5. A counterstain, safranin, was added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red. This was washed after 30 seconds with water. 6. Slides were dried in air and observation under microscope was done.

Isolation protocol (AREI)

Isolation protocol of Acute Respiratory and Enteric Infection diagnosis The term acute respiratory and enteric infections are used to combine two separate terms, Acute Respiratory Infection (ARI) and Enteric Infection (EI). The main focus of research is detection of pneumonia, disruption which often presents with acute respiratory tract infection. The source of this infection may be bacteria or virus. Amongst bacteria, Klebsiella pneumonia is most common causative organism, and amongst the viruses Haemophilus influenza and coronavirus cause infection. Most enteric infections cause diarrhea. Enteric infection by bacteria – salmonella, shigella, vibrio cholerae, E.coli cause fever too. • To detect ARI, 2 nasal swabs are taken from each person. • To detect EI, 5cc blood is taken from each person. In addition, to test for diarrhea, 2 rectal swabs or stool samples are taken from each person

Acute Respiratory Infection Protocol

Diarrhea Protocol

Enteric Fever Protocol

S. typhi and S. paratyphi store at -70°C

Gram positive Blood Born pathogens store at -70°C

ConclusionOn the whole, this internship as provided by the Department of Mathematics and Natural

Sciences, BRAC University was a useful experience. In addition, I was conversant with several

new people native to my field of study. I have learnt the different facets of working in a

professional environment especially in an industrial research laboratory. I was able to

comprehend several methodologies and techniques used in a research laboratory and the

differences they have in contrast to the experiments performed at educational laboratories.

The internship provided by the Department of Mathematics and Natural Science, BRAC

University was a great experienced for this education period. It gives to gather some practical

knowledge with some people who are from the same field just like me. In addition, it helps me to

be familiar with the new environment and some new experienced.

References 1. Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of

Vibrio cholera. Atlanta, Georgia: CDC; 1994.

2. World Health Organization. Manual for the laboratory investigations of acute enteric infections. Geneva: World Health Organization, 1987; publication no. WHO/CDD/83.3

3. Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of Vibrio cholera. Atlanta, Georgia: CDC, 1994.

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. Kay, BA, Bopp CA, Wells JG. Isolation and identification of Vibrio cholera O1 from fecal specimens. In: Wachsmuth IK, Blake PA, and Olsvik O., ed. Vibrio cholera and cholera:

molecular to global perspectives. Washington, DC: ASM Press; 1994: 3-26. Worksheet 53. 6. Guidelines for Biosafety in Teaching Laboratories (American Society for Microbiology

2012). 7. Basic laboratory procedures in clinical bacteriology / J. Vandepitte . . . [et al.].—2nd ed.)

WHO / 2003. 8. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth

Informational Supplement (January 2014).

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