A TECHNICAL REPORT ON

STUDENTS’ INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

UNDERTAKEN AT

NIGERIA INSTITUTE OF MEDICAL RESEARCH (NIMR)

6 EDMUND CRESENT, YABA, LAGOS MAINLAND 100001, LAGOS

BY
ADEPEGBA OLAMIDE MARY

U/21/MB/0032

SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES, MICROBIOLOGY

UNIT, COLLEGE OF NATURAL AND APPLIED SCIENCES, ODUDUWA
UNIVERSITY IPETUMODU, ILE IFE, OSUN STATE, NIGERIA.
IN PARTIALLY FULFILLMENT FOR THE AWARD OF THE DEGREE OF BACHELOR OF
SCIENCE (B.Sc.) IN MICROBIOLOGY

NOVEMBER 2024
 CERTIFICATION

This is to certify that ADEPEGBA OLAMIDE MARY with matriculation number

U/21/MB/0032 of the Department of Biological Sciences, microbiological unit, College of

Natural and Applied Science, Oduduwa University Ipetumodu Osun State participated in the

Students’ Industrial Work Experience Scheme (SIWES) at NIGERIA INSTITUTE OF

MEDICAL RESEARCH under the supervision of Mr Tobi. I also confirmed that this report is

only prepared for my academic requirement not for any other purpose. It has not been

presented for any awards degree in any institution.

MRS ABIOLA TAOLAT _________________________

PROJECT SUPERVISOR SIGNATURE AND DATE

Dr A. A OLAYINKA _______________________

HEAD OF DEPARTMENT SIGNATURE AND DATE

 DEDICATION

I dedicate this SIWES report at first to the almighty God, whose guidance, grace, favour and

love has been my source of energy and strength to go through day by day and has been my

anchor. I also dedicate this project to the Almighty God for wisdom, knowledge and

understanding He has given unto me. To my lovely family, I thank you for your support and

care in words and deeds. I thank you for your financial support and emotional support as

well. I greatly appreciate you in my life. Last but not least to my friend. I appreciate your

help, emotional support, academic support. May God bless you. I am so grateful for you all in

my life.
 ACKNOWLEDGEMENT

I extend my greatest acknowledgment to the Almighty God who has made all things well,

given me grace, favour and strengthen me during my industrial training. I also give thanks to

my family for their support in all areas of my training and constant encouragement and care.

My sincere appreciation goes to the staffs at the encouragement Nigeria Institute of Medical

Research, Mainland, Lagos, Lagos. Their training and collective support made me to have a

nice and positive experience during my time at the institute which helped me to gain more

experience about my course of study. My appreciation also goes to my supervisors in the

several departments which I was posted which are Mr Balogun, Miss Romoke, Miss

Temilade, Miss Blessing, Mr George, Miss Ibukun, Mr Ebenezer, Mrs Prisca, Miss Kehinde

for their individual role which sum up to the total knowledge I have attain during my training.

My special thanks to my supervisor Mr Tobi at Nigeria Institute of Medical Institute who

coordinated all my activities and departments which I was posted during the training period. I

acknowledge my supervisor Mrs Abiola Tailat for her immense supervision, I also

acknowledge my lecturers Dr A. A Olayinka, Dr Mrs Fakorede, Mrs Raji for their teachings. I

acknowledge my effort and immense hard work and persevering strength of the training

which allowed me to learn and participate in the training which increase my knowledge and

wisdom and helped me more in my field in microbiology.

 REPORT OVERVIEW

The students industrial work experience scheme was established to facilitate students’ ability

to see the practical aspects of what they have been learning in school, to help them to relate

with students of the same course of study from other institutions, to see how the activities of

being a staff is being done. To have a glimpse of the industrial life and what they course of

study looks like in the outside world.

My experience in NIGERIA INSTITUTE OF MEDICAL RESEARCH made my eyes

open to these I mentioned above, I was able to see the physical aspects of my course I was

able to see how issues occurs and is being resolved professionally. I was able to see how a

boss relate to his subordinate. I was able to have a responsibility

I was able to work in different lab where each lab is working different yet for the same

results, I was able to operate machines, equipment, relate with people of different beliefs,

thinking and culture. I learnt mainly on the technical aspect of virus. I was able to see people

with this virus physically, I saw their pain, struggle, I saw the reality of this virus. I was able

to see how to help them as well. And I learnt about laboratory safety as well. Generally, the

report contains the physical aspects of all I learned during my training which also include

other aspects. This technical report contains all I learnt during my training there, how real-life

situation were attended to and resolved.

Table of contents Pages
Certification
Dedication
Acknowledgement
Report overview
CHAPTER ONE
1.0 Introduction
1.1 Definition of SIWES
1.2 Background of SIWES
1.3 Objectives of SIWES.
1.4 Bodies involved in the managements of SIWES and their roles.
1.5 Description of Nigeria Institute of Medical Institute.
1.6 Mission of Nigeria Institute of Medical Institute.
1.7 Vision of Nigeria Institute of Medical Institute
CHAPTER TWO
2.1 Sample separation 1.
2.2 Procedure in sample separation 1.
2.3 Centrifuge.
 CHAPTER ONE

1.0 Introduction

1.1 Definition of SIWES

Student Industrial Work Experience Scheme (SIWES) is a skill training program designed to

prepare and expose of universities, polyethnic, college of agriculture and college of education

for the industrial work situation they are likely to meet after graduation. The essence of

SIWES is to bridge the gaps between theory and practice through the inculcation of

sustainable business and enterprise skills. SIWES was founded in 1973 by ITF (Industrial

Training Funds) to address the problem of tertiary institution graduates’ lack of appropriate

skills for employments in Nigerian industries. SIWES is done within three to six months. In

some schools SIWES is done at the end of second semester of either 300 or 400 levels. One

major disadvantage of SIWES is that in some cases, students lack proper supervision during

their SIWES placements.

1.2 Background of SIWES

The Students Industrial Work Experience Scheme (SIWES) is a unit under the Vice-

Chancellor’s Office. It was established in 2016. The Students Industrial Work Experience

Scheme (SIWES) is a skills training programme designed to expose and prepare students of

universities and other tertiary institutions for the Industrial Work situation they are likely to

meet after graduation. It is a cooperative industrial internship program that involves

institutions of higher learning, industries, the Federal Government of Nigeria, the Industrial

Training Fund (ITF), and the Nigerian Universities Commission (NUC). The industrial fund's

main motivation for establishing and designing the scheme in 1973/74 was launched against

this context. The ITF (Industrial Training Fund) organization decided to aid all interested
Nigerian students and created the SIWES program. The federal government officially

approved and presented it in 1974. SIWES was founded in 1973 by ITF (Industrial Training

Funds) to address the problem of tertiary institution graduates’ lack of appropriate skills for

employment in Nigerian industries.

1.3 Objectives of SIWES

Specifically, the objectives of the Students Industrial Work Experience Scheme (SIWES) are

to:

1. Provide avenue for Students in Institutions of higher Learning to acquire industrial

skills and experience in their course of study.

2. Prepare Students for the industrial work situation they are to meet after graduation.

3. Expose Students to work methods and techniques in handling equipment and

machinery that may not be available in their Institutions.

4. Make the transition from school to the world of work easier, and enhance Students

contacts for later job placement.

5. Provide Students with an opportunity to apply their knowledge in real work situation

thereby bridging the gap between theory and practice.

6. Enlist and strengthen Employers involvement in the entire educational process and

prepare Students for employment after graduation

1.4 Bodies involved in the management of SIWES programme and their roles

The Federal Government of Nigeria,

1. The Industrial Training Fund (ITF),

2. National Universities Commission (NUC),

3. National Board for Technical Education (NBTE),

4. National Commission for Colleges of Education (NCCE),

5. Institutions of Higher Learning;

6. The Employers of Labour

The bodies have specific roles assigned to them in the administration and management of

SIWES.

FEDERAL GOVERNMENT OF NIGERIA

1. Provide adequate funds to the Industrial Training Fund through the Federal Ministry

of Industry, Trade & Investment for the Scheme.

2. Make it mandatory for all Ministries, Companies and Parastatals to offer places for

the attachment of Students in accordance with the provisions of Decree No. 47 of

1971 as amended in 2011. The relevant provisions of the amended act are as follows:

Section 7A (1) (b) stipulates as follows:

Shall Accept Students for Industrial Attachment Purposes

The Decree under section 7A (2) stipulate penalties in default of section 7A (1)(b).

Section 7A (2)

“Any employer who is in breach of the provision of the sub-section (1) of this section shall be

guilty of an offence under this Act and liable to conviction:

In the case of a corporate body, to a fine of Five Hundred Thousand Naira (N500,000.00) for

the first breach and One Million Naira (N1,000,000.00) for subsequent breach; and
In the case of Chief Executive, Secretary, or other Principal Officers of the company, to a fine

of Fifty Thousand Naira(N50,000.00) or two years imprisonment for the first breach and

three years’ imprisonment without option of a fine for each subsequent breach.

THE INDUSTRIAL TRAINING FUND

1. Formulate policies and guidelines on SIWES for distribution to all SIWES

participating bodies, institutions and companies involved in the Scheme;

2. Regularly involve in organizing orientation programmes for Students prior to their

attachment;

3. Receive and process Master and Placement Lists from the Institutions through their

Supervisory Agencies (NUC, NBTE & NCCE);

4. Supervise and Monitor Students on Industrial Attachment;

5. Disburse Supervisory and Students allowances by e-payment.

6. Organize Biennial SIWES National Conference and SIWES Review Meetings;

7. Provide insurance cover for Students on attachment;

8. Provide logistics and materials necessary for effective administration of the Scheme.

9. Ensure the visitation (tours) of ITF Officers to the Supervising Agencies, Institutions,

Employers and Students on Attachment;

10. Provide information on companies for attachment and assist in the industrial

placement of students;

11. Continuously review and carry out research into the operation of the Scheme;

12. Vet, process and verify Students’ logbooks and ITF Form 8.

1.5 Description of Nigeria Institute of Medical Research

The Nigerian Institute of Medical Research (NIMR) in Yaba, Lagos state, Nigeria is a

medical research institute established by the Federal Government of Nigeria through the
research institute establishment act of 1977, to promote National health and developments.

NIMR focuses on scientific area of research in Biochemistry and Nutrition, Virology

Vaccinology, Immunology, Health system and policy research, Reproductive, Maternal and

Childhood diseases Research, Clinical Science, Microbiology, Molecular biology

Biotechnology and public Health, with studies that focus on diseases of greatest public health

importance in the country. These include: Malaria, HIV/AIDS Tuberculosis, Hepatitis,

Schistosomiasis, Helicobacter Pylori, and Typhoid.

On 18 September 2020, NIMR unveiled the first SARS-CoV-2 Isothemal Molecular Assay

(SIMA) kit in Nigeria to boost COVID-19 testing capacity.

There are several centres in NIMR which include Centre of Human Virology and Genomics

(CHVG), Centre of Infectious Diseases and Research (CIDR), Centre for Tuberculosis

Research (CTBR). These centres have different laboratories.

1.6 Organogram of the organisation.

 Figure 1.0 Organogram of the organisation.

1.6.1 Structure of the department of the microbiology

The department of Microbiology at the Nigerian Institute of Medical Research is made up of

three centres, as well as a Production Unit. The centres are as follows:

Centre for Human Virology and Genomics (CHVG)

Centre for Tuberculosis Research (CTBR)

Centre for Infectious Disease Research (CIDR).

FMoH

Governing board

Clinical specialist Quality management

Director general
system

Infection control Head of department

Equipment

Monitoring and evaluation Training

Centre for human Centre for tuberculosis Centre for infectious disease
virology and genomics research research

Research Services Research Services Research Services

1.8 Mission of Nigeria Institute of Medical Research

To conduct research into diseases of public health importance in Nigeria and develop

structures for the dissemination of research findings while providing the enabling

environment and facilities for health research and training in cooperation with the federal and

state ministries of health and in collaboration with universities, allied institutions and

organized private sector nationally and internationally.

1.9 Vision of Nigeria Institute of Medical Research

To be the leading national institution building capacity for basic, applied and operational

research for the promotion of national health and development.

1.10 Core values

The core values are honesty, integrity, leadership, excellence, respect, fairness, dignity,

teamwork, responsibility, relevance, innovation, hard work, fair reward and recognition,

accountability and transparency, communication-internal and external, equity.

 CHAPTER TWO

2.0 Description of works.

2.1 General laboratory equipment and instrument

CENTRIFUGE.

Centrifuge is a laboratory equipment that separates particles of different densities or sizes

from a mixture by applying centrifugal force. This force causes particles to sediment or float

according to their densities, allowing for efficient separation and isolation of various

components.

REFRIGERATOR

Refrigerator is an equipment used in the laboratory to preserve samples and prevent the

growth of micro-organisms. The refrigerator helps provide optimum environment for

materials to be preserved. The samples are stored according to the day it was separated. In the

refrigerator, the separated samples and the remaining blood after separation are kept there.

COBAS 8800 and COBAS 6800

These are machines which are robot-like and are the major equipment used in this laboratory.

These are the machines used to count the amount of virus present in the samples. This

machine comprises of four modules which are sample supply module, sample transfer

module, sample processing module 1&2, analytical module. Each module has compartment

where consumables are given to the machine.

BIOSAFETY CABINET
This is an equipment used in dispensing samples from one tube to another. It protects the

environment, personnel and the sample. It helps to maintain the quality of the material bring

worked on.

AUTOCLAVE

The autoclave is an effective equipment used for steam sterilization at pressures above the

atmospheric pressure. Thus, it is possible to steam at higher temperature then the boiling

point, which a lot of microorganisms cannot withstand. Autoclaving is the most effective

method for sterilizing culture media.

INCUBATOR

The incubator is mainly used to incubate culture media as microbes have different optimum

temperatures for growth and reproduction. The temperature of an incubator can be set to the

preferred temperatures.

MICROSCOPE

Microscopes are used in viewing specimens that are relatively very small in size, they are

used to view the cellular structures of organ, germs, and bacteria. They play a very important

role in the laboratory for the tissues and organisms which are too small to be seen clearly with

naked eye. Types of microscopes include simple Microscope- Microscope made up of single

lens. Compound Microscope -Microscope made up of two sets of lenses. Microscope can also

be classified into Electron Microscope, Light Microscope, Optical Microscope, Confocal

microscope.

WEIGHING BALANCE

This is a delicate instrument used for weighing essential, reagent, stains and culture media

that requires adequate weighing.

WIRE LOOP

It is made up of a thick metallic lower part and a straight thin upper metallic part curved into

a small circle usually made up of platinum. Wire loop is used generally for inoculating

samples and picking colonies sterilized by flaming red hot before, during and after use. It is

always better to use the sides of the loop rather than the apex during inoculation.

GLASS SLIDES

Used for preparation of slides for microscopy. Sterilization is by flooding with alcohol and

flaming off excess alcohol.

PETRI DISH

Used for the preparation of culture media. It is usually bought sterilized. The disposable type

cannot be used a second time while the glass ware type can be reused when sterilized by

autoclaving.

FORCEPS

A pair of forceps is a metallic object used for handing hot object or contaminated materials. It

is sterilized by flaming till it is red hot.

COVER SLIPS

This is use for covering wet smears of preparations. It is sterilized by flooding with alcohol

and flaming off excess alcohol.

PETRI DISH

It is used for the preparation of culture media. It is usually bought sterilized. The disposable

type is used in the laboratory.

Others include:
Needles, Syringe, Sterilized bottle, Gram positive and negative sensitivity kit, Swab stick,

Surgical blades, Fluorescence microscope, Hot plate (dryer), Hand gloves, Capillary tubes for

measuring PCV, Anaerobic jar, Test tubes, pipette, beakers.

2.2 Centre of Human Virology and Genomics

Centre of Human Virology and genomics is one of the three centres in the department of

microbiology where both research and services related to viruses are done.

Centre of human virology and Genomics (CHVG) is an ISO 15189 accredited laboratory.

This department has been involved in the diagnosis of disease such as yellow fever, Lassa

fever. In the spirit of providing social corporate responsibility, in 2023 they expanded the test

menu to include Hepatitis surface antibody (HbsAb). Also, making it more convenient for

travellers by expanding the test menu to include Varicella, Rubella, Measles, Mumps and

other assays needed for immigration purposes. There are notable commemoration days which

include world blood donor day, world hepatitis day, customer service week, world AIDS day.

2.3 SAMPLE SEPARATION 1

This is a laboratory where samples are being separated as the name implies. There are

materials and equipment used in this laboratory including centrifuge, refrigerator, lab coat,

cryovial tubes, EDTA bottles, gloves, biohazard bin, refuse bin, bleach, methylated spirit,

SOP, table cover, wipes, samples, Styrofoam, samples racks, air conditioner, thermometer,

cold chain

The laboratory is divided into two areas which are clean area and dirty area
 Figure 2.0 Images showing clean and dirty area.

2.3.1. PROCEDURE IN SAMPLE SEPERATION 1

Samples are taken from the NIMR clinic/phlebotomy by the staff /IT/interns by checking a

form containing the identity of the sample given to them by the professional in the clinic and

any sample without a form is not taken. These samples are blood drawn from patients. Then

the samples are arranged in a cold chain to maintain the temperature. It is then brought to the

laboratory for separation. The sample identity which are the samples’ identity number, patient

initials, test being carried out and date are written down inside a register.
Figure 2.1
Image of
blood
samples
after
Figure 2.2 Image of pipetting process using Pasteur pipette
The next step is the centrifugation. In this process, the EDTA bottles containing the samples

will be arranged in the centrifuge in an ascending manner and then close and the start button

will be pressed before the start button is pressed the time is set to 20 minutes and 4.4 rpm

(revolution per minute). Then the centrifuge starts to work.

Fig 2.3: Centrifuges

2.3.2 Centrifuge.

Centrifuge is a laboratory equipment that separates particles of different densities or sizes

from a mixture by applying centrifugal force. This force causes particles to sediment or float

according to their densities, allowing for efficient separation and isolation of various

components.

2.3.3 Refrigerator

Refrigerator is an equipment used in the laboratory to preserve samples and prevent the

growth of micro-organisms. The samples are stored according to the day it was separated.

In the refrigerator, the separated samples and the remaining blood after separation are kept

there.
Figure 2.4 Refrigerator
2.3.4 Data collection

The samples have forms attached to it by which the staff will check before collecting the

samples at the clinic and at phlebotomy and samples without forms or inaccurate details will

not be accepted. Each sample will be documented in a register book as well.

2.3.5 Materials and equipment

Materials and equipment used in sample separation one are as follows Styrofoam rack,

centrifuge, refrigerator, biohazard bins, refuse bins, EDTA tubes, Pasteur pipette, gloves,

laboratory coats, bench cover, markers, register books,

2.4 Viral load

2.4.1 Definition

This is a laboratory in Centre of Human Virology and Genomics where the amount of virus

present in a plasma sample is counted by the use of a machine called Cobas 8800 and Cobas

6800 machine. The machine can accept 93 samples and 3 controls at a time.

2.4.2 Cobas 8800 and Cobas 6800

These are machines which are robot-like and are the major equipment used in this laboratory.

These are the machines used to count the amount of virus present in the samples. This

machine comprises of four modules which are sample supply module, sample transfer

module, sample processing module 1&2, analytical module. Each module has compartment

where consumables are given to the machine.

Figure2.5 Cobas 8800
Figure 2.6 Cobas 6800
2.4.2.1 Samply supply module

This module comprises of four lanes which are input lane, output lane, error lane and

emergency lane. The input lane is where the samples are arranged into the machine and

entered into the machine to be processed. The output lane is where the samples are brought

out after being processed and counted. The error lane is where the samples are brought out

during the processing as a result of presence of clots, of inaccurate barcoding or insufficient

volume of sample. The emergency lane is used when a sample needs to processed

immediately. This module can be detachable which means it can work on its own

2.4.2.2 Sample transfer module

This is the module where the machine takes 500 microlitres of the samples and transfer it into

the Cobas omni processing plate using the omni pipette and tips avoiding which is the second

stage of pipetting. The compartment under the sample transfer contains 2 positive controls, 1

negative control, processing plate which contains 48 wells. The machine contains one transfer

module.

2.4.2.3 Samply processing module

This is the module where the samples are transferred from the processing plates to

amplification plates. In the module there are three stages requires which are lysis, washing,

dilution and extraction. The cells are first lysed with the Cobas omni lysis to open it the it is

washed with the omni wash to wash off other organelles of the cells apart from the nuclei

acid thereby remaining the genetic material. The Cobas omni MGP (Magnetic Glass Particle)

reagent binds to the nuclei acid to prevent it from being washed away during the washing, it

is then diluted. The main action happening in the sample processing module is RNA
extraction which finally give eluents (the purified RNA). The machine has two processing

modules.

2.4.2.4 Analytical module

This is the module where the Polymerase Chain Reaction (PCR) takes place where the DNA

is replicated to produce extra DNA copies which is done by action of heat. This is done in

real-life but automated process. The machine contains four analytical modules.
Figure2.7 Sample transfer module
Figure 2.8 Sample processing
module.
2.4.2.4.1 Polymerase Chain Reaction

Polymerase chain reaction is the process by which multiple copies of DNA is made. There are

different types of polymerase chain reaction which are convectional PCR, real-time PCR,

Reverse transcription PCR. Reverse transcription PCR is done for HIV viral load.

2.4.2.4.1.1 Denaturation

The DNA is subjected to heat at 95⁰C which breaks the double helical structure to two single

strands.

2.4.2.4.1.2 Annealing

The primer then attaches to the DNA at the origin. This takes place at 55-65⁰C.

2.4.2.4.1.3 Extension

The taq polymerase then add complementary nucleotides to the rest of DNA from 3 prime.

2.4.3 Materials and compartments needed in the Cobas 8800& Cobas 6800

Sample racks, Sample tray, Omni-short pipette tip, Omni-long Pipette tip, Omni Cobas wash

reagent, Omni negative control Kit, Omni positive control kit, Omni amplification plate,

Omni MGP reagent, Omni lysis reagent, Omni special diluent.

2.4.3.1 Materials needed and their functions.

2.4.3.1.1. Sample racks: It contain five secondary tubes.

2.4.3.1.2. Sample tray: It contains several samples racks

2.4.3.1.3. Omni-short pipette tip: It is used during the aliquoting and precise reagent

addition steps. This includes transferring small and accurate volumes of reagents and samples
for reactions or PCR setup. The short tip is essential for tasks requiring high precision in

small volumes, such as adding primers, enzymes, or other small reagent volumes.

2.4.3.1.4. Omni-long Pipette tip: Employed during initial sample loading and washing

stages, where larger volumes of liquids need to be handled. The long tip is used to transfer

samples from the input racks to the processing plates and to perform washing steps where

larger volumes of wash buffers are required.

2.4.3.1.5. Omni Cobas wash reagent: This reagent is used to clean and decontaminate the

Cobas system’s components between samples, ensuring that there is no cross-contamination

and that the system in optimal working condition.

2.4.3.1.6. Omni Negative Control Kit: This kit contains a negative control, which is used to

ensure that the test runs are free from contamination and that the reagents and processes are

functioning without producing false positives.

2.4.3.1.7. Omni Positive Control Kit: This kit includes a positive control, which is used to

verify that the system is functioning correctly and can detect the target analyte. It helps in

confirming the accuracy and reliability of the test results.

2.4.3.1.8. Omni Amplification Plate: This plate is utilized in the amplification step of the

testing process, where the target DNA or RNA is amplified for detection.

2.4.3.1.9. Omni MGP Reagent: The Magnetic Glass Particle (MGP) reagent is used for the

extraction and purification of nucleic acids from the samples. The magnetic particles help in

binding and separating the nucleic acids during the extraction process.

2.4.3.1.10. Omni Lysis Reagent: This reagent is used to break open cells and release their

contents, including nucleic acids, which are then available for extraction and subsequent

analysis.
2.4.3.1.11. Omni Special Diluent: This diluent is used to prepare samples and reagents to the

appropriate concentrations needed for accurate and reliable testing.

2.4.3.2 Compartments of the machine

1 sample supply module, 1 sample transfer module, 2 sample processing module, 4 analytical

modules.

2.4.4 Comparison between Cobas 8800 &Cobas 6800.

Cobas 8800 and Cobas 6800 have one sample supply module and sample transfer module,

Cobas 8800 has two processing module and four analytical modules while Cobas 6800 has

one two processing and two and analytical module.

2.4.5 Aliquoting section.

This is the section where the manual transfer of plasma from the cryovial tubes into the

secondary tubes. This is done in a biosafety cabinet with the personnel wearing lab coat and

gloves. This is done to protect the environment, personnel and the samples. Aliquoting is

done to have a consistent volume of plasma samples and to remove any clot present in the

plasma sample.

2.4.5.1 Materials needed for aliquoting process.

Biosafety cabinet, gloves, micropipette, pipette tips, secondary tubes, cryovial tubes, discard

jars.

2.4.5.2 Procedure for aliquoting.

Personnel wearing a lab coat and gloves with cover shoe will take 500 microliters of the

plasma from the cryovial tubes into the secondary tubes in an ascending order
2.5 Controls.

The controls used in the Cobas machine are to ensure quality. Once the controls fail, the

running samples will fail. There are positive and negative controls. The positive controls are

of two types namely high positive control and low positive control. The high positive control

is for samples having more than 10,000 viral load while the low positive control is for

samples having less than 20 viral load.

2.6 CD4

CD4 laboratory is a laboratory where CD4 test is carried out. CD4 are T-helper cells which

are immune cells, they protect the body against infection. People with HIV/AIDS have

compromised immunity/ immunity deficiency which makes their immune cells weak.

Therefore, they have low amount of CD4 cells to fight against infections in their body. CD4

cells are the target of HIV in the body.

2.6.1 Materials and equipment used in CD4 laboratory

Cy flow counter, micropipette, micropipette tips, Rohrer tubes, Dyna mixer, decontamination

solution, cleaning solution, sheath fluid, racks.

2.6.2 Procedure.

2.6.2.1. The CD4 antibody, dilution buffer and count check beads green must be at room

temperature before use

2.6.2.2. The samples which is the blood of the patient is collected in the EDTA bottle.
2.6.2.3. It is then arranged and mixed on the dyna mixer for 5 minutes to prevent from

settling down.

2.6.2.4. Using the micropipette and pipette tips 20 microlitre of the monoclonal antibody are

pipette into the Rohrer tubes.

2.6.2.5. Then 20 microlitre of the blood sample is added to it. It is then shaken together.

2.6.2.6. It is then incubated in the dark for 15 minutes.

2.6.2.7. 800 microlitre of no-lyse buffer was then added to the bottle and shaken gently

together. The tube must not touch the electrode.

2.6.2.8. The speed of the Cyc flow counter was then set to 18 and the machine was started.

2.6.2.9. Pre-run appear on the machine first, then run, then count and finally ready. The sound

of the machine must stop before the printing the result.

2.6.2.10. Then information is printed out in a paper showing the graphical representation of

the machine spinning.

 Figure 2.9: Decontamination solution,
cleaning solution, sheath fluid
 Figure 2.10: Cyflow counter screen.

2.6.3 Internal quality control.

2.6.3.1. Select one sample (range finding sample) prepare and store the aliquots at 2-8 degree

Celsius

2.6.3.2. Assay the prepared samples next day. One aliquot in the morning and the second

during the all and compare with the previous day values.

Calculation of IQC =Actual value-observed value/ actual value times 100%

The control is said to have passed if the compared values are within +/-10% of the previous

values.

2.6.4 Daily start up cleaning of the Cyc flow counter

2.6.4.1. Run 1600 microlitre of decontamination solution on the machine

2.6.4.2. Run 1600 microliter of cleaning solution on the machine.

2.6.4.3. Run 1600 microliter of the sheath fluid on the machine.

2.6.4.4. Run another 1600 microliter of sheath fluid on the machine and it leave it there.

The acceptable background count cell after daily start -up procedure and after every clean is

less than or equal to 5 cells per microliter. If it displays higher than five cells, the machine

needs to cleaned.

2.6.5 Shut down cleaning procedure

2.6.5.1. Run 1600 microliter of decontamination solution on the machine

2.6.5.2. Run 1600 microlite of cleaning solution on the machine

2.6.5.3. Run 1600 microliter pf sheath fluid on the machine. Press cancel after 1 minute.

2.6.5.4. Leave the tube with the sheath fluid attached to the sample port.

2.6.5.5. Allow the machine to stabilize for 10 minutes.

2.6.5.6. Switch off the machine.

2.6.6 Performing the measurement

2.6.6.1. Settings ---- Load script----CD4 script.

2.6.6.2. Insert the sample into the sample port.

2.6.6.3. Click START to start the measurement.

2.6.6.4. PRERUN appear on the machine-creates a temporary, fast sample flow.

2.6.6.5. MEASURE-the particles are measured and counted

2.6.6.6. LEVEL COUNTING-a certain volume get measured and counted.

2.6.6.7. FLUSH- an automatic internal cleaning cycle get performed

2.6.7 Calibration

For calibration, the CCBG (Count Check Beads Green) is being used. The LOT specific

concentration indicated on the label of the CCBG bottle is compared with the result. Check if

the result is within +/-10% range of the LOT specific concentration on the CCBG bottle. If

the result is below +/- 10% range, the Cyc flow counter is ready for further analysis.

2.6.7.1 Calibration procedure

2.6.7.1.1. Bring out the CCBG out of the refrigerator to attain room temperature

2.6.7.1.2. Shake, for 2 minutes and pipette 850 microlitre of the CCBG into the sample tube

(Rohrer tube).

2.6.7.1.3. Then plug into the sample port and press the START bottom till it is successfully

then print out.

NOTE: The CCBG is done weekly.

2.6.8 CD4 result interpretation.

The CD4-T cells (prominent peak on the right) can be clearly separated from the CD4

monocytes (left peak of weaker inflorescence intensity).

2.6.9 CD4 result reading

Any value above 200 cells per microliter is a healthy amount while panic value which are less

than 200 cells per microliter indicates that the patients is progressing towards AIDS or

already has AIDS. Panic value is released within 48 hours of sample run to validation unit,

authenticated and documented.

2.6.10 Visitect CD4

2.6.10.1. Write the patient ID on the device.

2.6.10.2. Measure 30 microliter if the blood sample into well A and wait for 3 minutes.

2.6.10.3. Add a drop of the buffer solution into the well A containing the blood sample and

wait for 17 minutes. Hold the buffer bottle vertically 1xm above well B. Carefully add 3

drops of buffer to well B allowing each drop to absorb into the well before adding the next

drop.

2.6.10.4. Wait for 20 minutes

2.6.11. Precautions regarding Visitect CD4

2.6.11.1. Allow the test kit to come to operating temperature (15-35 degree Celsius). Check

expiry on foil pouch.

2.6.11.2. Discard the sampling device/disposable tip into a sharp bin

2.6.11.3. After the test is completed, interprets the results within 5 minutes. Refer to examples

of results on reverse.

2.6.11.4. The control line and reference (200) line must be present when reading the test

results for the test to be valid.

2.6.12Interpretation of results

2.6.12.1. Test (T) equal to reference (200) line=Below reference.

2.6.12.2. Test(T) line missing=Below reference

2.6.12.3. Test(T) line lighter than reference (200) line=Below reference

2.6.12.4. Test(T) line darker than reference (200) line= Above reference

2.6.12.5. Reference (200) line missing = Invalid, repeat this test

2.6.12.6. Control (C) line missing= Invalid, repeat this test.

2.7 Phlebotomy.

This is a department where the drawing of blood from patients, documentation, payments

take place. The process of drawing blood is done by the phlebotomist.

2.7.1 Materials needed in phlebotomy

Tourniquet, EDTA bottles, plain bottles, needles, syringe, needle holder(vacutainer),

disposable bin, table cover, kidney dishes, cotton wool, spirit,70% ethanol, lab coat, gloves.

2.7.2 Request Form Identification

2.7.2.1. Patient’s identification which include gender, date of birth, location, contact details

and unique identification and unique identifier (laboratory number or clinic number)

2.7.2.2. Name other unique identifier of clinician/healthcare provider as well as the

destination of the report and contact details.

2.7.2.3. Type of primary sample

2.7.2.4. Examination(s) requested

2.7.2.5. Relevant clinical information/diagnosis for examination performance and for results

interpretation purposes.

2.7.2.6. Date & time of primary sample collection.

The request form is on paper but can be sent electronically to patient outside Lagos.

2.7.3 Procedure for Verbal Requests and Additional Examination.

2.7.3.1. Recipient first confirms that the sample collected will be adequate for the additional

test.
2.7.3.2. Ensure that the request came within the time limit of the particular test requested

Haematology/CD4- 24 hours

Serology/chemistry- 7 working days

All PCRs -14 working days

2.7.3.3. Such requests are documented at the reception with appropriate remark on the request

form.

2.7.3.4. Such information should be communicated to relevant benches/personnel at

analytical and post analytical for necessary action.

2.7.3.5. Once results have been released, verbal request will not be acceptable because

samples would have been discarded.

2.7.4 Instruction for Pre-collection Activities

2.7.4 1. All new clients are given CHVG request form to fill in order to have their complete

information and the test they are requesting, while the old client with request form is required

to write phone number and current address on the request form they came with, in order to

update their data.

2.7.4.2. Note that CHVG request form is given to any patient/client who comes with request

form (whether old or new patients).

2.7.4.3. All columns in the request form are to be completed except for where is not

applicable.

2.7.4.4. Patient confidentiality- the patient information should not be disclosed to authorised

personnel. The patients’ codes and initial are used on the container instead of their names.
2.7.4.5. In CHVG, patients are attended to at the reception but physically challenges

challenged ones are attended even at the front of the lab.

2.7.4.6. Greet each patient/client that comes in and call them on the name on their card or

request form and confirm from them the yest they are to do.

2.7.4.7. Explain the procedure to be performed to the patients/client. This types and amount

of samples to be collected with a description of the container as well as the additive and

soliciting for their cooperation.

2.7.4.8. Verify that the patient’s pre-examination requirement (fasting for FBS).

2.7.4.9. Inform the patient of when and how the results will be sent to them.

2.7.4.10. Inform the patient of when and how the results will be sent to them. Obtain verbal

consent from patients/guardian by asking permission to proceed with the procedure.

2.7.4.11. Personnel from information desk verifies with phone number of one randomly

patient seated at the reception waiting area by calling the phone number the patient wrote on

their request form.

2.8 Serology

In this laboratory, each test lit comes with different manual so one has to read the manual as

the process is going on.

2.8.1 Tests in serology

These tests are numerous but we will discuss the following, HIV Ag &Ab, HBe Ag &Ab,

HIV confirmation test, HCV Ab, HBs Ag

2.8.1.1 HIV confirmation test

2.8.1.1.1. 2 drops of the plasma sample was drop on the HIV test kit
2.8.1.1.2. In this test, if the line shows only on the control line it is negative, if the control and

then one 1 or 2 then it is positive.

This test is done to conform the HIV status of the patient, then to check if a positive patient

has either HIV 1 or HIV 2.

2.8.1.2 HIV Ag & Ab

2.8.1.2.1. Carefully establish the sample distribution and identification plan.

2.8.1.2.2. Prepare the diluted washing solution R2 and conjugate 2 working solution

(R72+R7b)

2.8.1.2.3. Take out from the protective packing the support frame and the necessary number

of strips(R1).

2.8.1.2.4. Distribute in the well in the following order, without prior washing of the plate:

2.8.1.2.4.1. 25 µl of conjugate 2(R6) in each well

2.8.1.2.4.2. 75 µl of HIV Ag Positive control (R5) in well A1

2.8.1.2.4.3. 75 µl of HIV Ab Positive control (R4) in well B1.

2.8.1.2.4.4. 75 µl of negative control (R3) in well C1, D1 and E1.

2.8.1.2.4.5. 75 µl of specimen 1 in well F1

2.8.1.2.4.6. 75 µl of specimen 2 in well G1.

2.8.1.2.5. When possible, cover the microplate in a thermostat-controlled water-bath or

microplate incubator at 35⁰C

2.8.1.2.6. Incubate the microplate in a thermostat-controlled water-bath or microplate

incubator at 37⁰C ± 1 degree for 1 hour ±4 minutes

2.8.12.7. Then the washing process, the washing is a total of a minimum of 3 washes

2.8.1.2.8. Quickly dispense 100 µl of conjugate 2 (R7a+R7b) into each well within the plate.

The conjugate must be shaken gently before use. Cover of it is possible with a new adhesive

film and incubate for 30 minutes ± 4 minutes at room temperature.

2.8.1.2.9. If necessary, remove the adhesive film, empty all the wells by aspiration and wash a

minimum of 5 times

2.8.1.2.10. Prepare the enzymatic development solution (reagent R8+R9)

2.8.1.2.11. Quickly distribute 80 µl of prepared enzymatic development (R8+R9) in all the

wells. Allow the reaction to develop in the dark for 30 minutes ±4 minutes at room

temperature (18-30 Celsius). Do not use adhesive during this incubation

2.8.1.2.12. Add 100 µl of the stopping solution (R10) using the same sequence and the rate of

distribution as for the development solution.

Pink (for negative samples) or blue (for positive samples) fades from the wells which

becomes colourless (for negative samples) or yellow (for positive samples) after adding

stopping solution.

2.8.1.2.13. Carefully wipe each plate bottom, wait at least 4 minutes after stopping solution

addition and within 30 minutes of stopping the reaction, read the optical density at 450/620-

700nm using a plate reader.

2.8.1.2.14. The enzymatic development was prepared and homogenized.

1 ml of R9+10 ml of R8 then homogenized

2.8.1.3 To get the results

2.8.1.3.1. Calculate the mean of the three negative control

2.8.1.3.2. Then, calculate the cut-off (CO) value

Cut off value=Mean of the negative control+0.200

2.8.1.3.3. To calculate the ratio of the optical density of the sample against the cut-off value

2.8.1.3.4. Value less than 1 =negative, greater than 1=positive.

2.8.1.3. HBe Ag &Ab

2.8.1.3.1. HBe Ag-Hepatitis Be Antigen

HBe Ag is associated with hepatitis B virus replication and the presence of infectious Dane

particles in the blood. Sine, HBe Ag is a specific sign of infectivity, the presence of anti HBe

Ag antibodies (HBe Ag) in the blood is recognised to be clinical sign of recovery from

infection to convalescence. The determination of these two analytes in samples from HBV

patients has become important for the classification of the phase of illness and as a prognostic

value in the follow up of infected patients.

2.8.1.3.1.1. Principle of HBe Ag

HBe Ag, if present in the sample, is captured by a specific monoclonal antibody in the 1 st

incubation. In the 2nd incubation, after washing, a tracer composed of a mix of two specific

anti HBe Ag monoclonal antibodies labelled with peroxidase (HRP) is added to the

microplate and binds to the captured HBe Ag. The presence of HBe Ag in the sample is

determined by means of cut-off value that allows for the semi qualitative detection of the

antigen.

2.8.1.3.1.2 Procedure of the HBe Ag test

2.8.1.3.1.2.1. Place the required number of strips and plastic holder and carefully identify the

wells for controls, calibrator and samples

2.8.1.3.1.2.2. Leave A1 well empty for blanking purposes.

2.8.1.3.1.2.3. Pipette 100 µl of negative control in triplicate, 100 µl of the antigen calibrator

in duplicate and then 100 µl of the antigen positive control in single.

2.8.1.3.1.2.4. Then dispense 100µl of samples in the proper wells.

2.8.1.3.1.2.5. Check for the presence of samples in wells by naked eyes

2.8.1.3.1.2.6. Incubate the microplate for 60 minutes at +37 ⁰C.

Do not touch the inner surface of the well with the pipette tip and not to immerse the top of it

into samples or controls. Contamination might occur.

2.8.1.3.1.2.7. When first incubation is finished, wash the microwells as previously described.

2.8.1.3.1.2.8. Dispense 100 µl of enzyme conjugate in all wells, except for A1, used for

blanking operations.

2.8.1.3.1.2.9. Check that the reagent has been dispensed properly and then incubate the

microplate for 60 minutes at +37⁰ C

2.8.1.3.1.2.10. When the second incubation is finished wash the microwells as previously

described.

2.8.1.3.1.2.11. Pipette 100 microliter chromogen/substrate into all wells. A1 included. Do not

expose to strong direct light as a high background might be generated

2.8.1.3.1.2.12. Incubate the microplate protected from light at room temperature (18-24 ⁰C)

for 20 minutes. Wells dispersed with positive control and positive samples will turn from

clear to blue.

2.8.1.3.1.2.13. Pipette 100 µl of sulphuric acid into all the wells using the same pipetting

sequence as in step 11. Addition of the stop solution will turn the positive control and positive

samples from blue to yellow.

2.8.1.3.1.2.14. Measure the colour intensity of the solution in each well, as described.

2.8.1.3.1.3 To get result for the HBe Ag test.

2.8.1.3.1.3.1. Average of the three negative controls

2.8.1.3.1.3.2. Then, cut off=average of negative+0.100

2.8.1.3.1.3.3. Positive control.

2.8.1.3.1.4 HBe Ag result interpretation.

2.8.1.3.1.4.1. Sample result/cut-off=less than 0.9(means that the result is negative)

2.8.1.3.1.4.2. Sample result/cut-off=0.9-1.1(means the result is equivocal)

2.8.1.3.1.4.3. Sample result/cut-off=greater than 1.1(means that result is positive)

2.8.1.3.2 HBe Ab

Antibodies, if present in the sample compete with a recombinant HBe Ag preparation for a

fixed amount of an anti HBe Ag antibody coated on the microplate wells. The competitive

assay is carried out in two incubations, the first with the sample and rec HBe Ag and second

with a tracer composed of two anti HBe Ag monoclonal labelled with peroxidase (HRP)

antibodies in the sample and its activity by adding the Chromogen/substrate in the 3 rd

incubation.

2.8.1.3.2.1 Procedure of HBe Ab test

2.8.1.3.2.1.1 Place the required number of strips in the plastic holder and carefully identify

the well controls, calibrator and samples.

2.8.1.3.2.1.2 Leave A1 well empty for blanking purposes.

2.8.1.3.2.1.3 Pipette 50 µl of the negative control in triplicate, 50 µl of antibody calibrator in

duplicate and then 50 µl of the antibody positive control in single.

2.8.1.3.2.1.4 Then dispense 50 µl of samples in the proper wells.

2.8.1.3.2.1.5 Check for the presence of samples in wells by naked eye.

2.8.1.3.2.1.6 Dispense then 50 µl of HBe antigen in all the wells except for A1.

2.8.1.3.2.1.7 Incubate the microplate for 60 minutes at + 37 ⁰C.

2.8.1.3.2.1.8 When the first incubation is finished, wash the microwells.

2.8.1.3.2.1.9 Finally proceed as described for the HBe Ag assay from point 8 to the last one.

2.8.1.3.2.2 To get results for the HBe Ab test

2.8.1.3.2.2.1 Average of the three positive controls

2.8.1.3.2.2.2 Positive control

2.8.1.3.2.2.3 Average of the positive controls+ positive divide by 3

2.8.1.3.2.3 HBe Ab result interpretation

2.81.3.2.3.1 Cut off/Sample =less than 0.9 (means the result is negative)

2.8.1.3.2.3.2 Cut off /Sample =0.9-1.1 (means the result is equivocal)

2.8.1.3.2.3.3 Cut off/ Sample = greater than 1.1 (means the result is positive)

2.8.1.4 HCV Ab (Enzyme Immunoassay for the determination of anti-Hepatitis C

virus antibody in Human serum and plasma for ‘in vitro’ diagnostic use only.

2.8.1.4.1. Procedure of HCV Ab test.

2.8.1.4.1.1. Place the required number of microwells in the microwell holder. Leave the 1 st

well empty for operation of blanking.

2.8.1.4.1.2. Dispense 200µl of negative control in the triplicate, 200µl in duplicate and 200µl

positive control in single in proper wells. Do not dilute controls and calibrator as they are pre-

diluted ready to use.

2.8.1.4.1.3. Add 200µl of sample diluent (DILSPE) to all the sample wells, then disperse 10

µl sample in each properly identified well. Mix gently the plate, avoiding overflowing and

contaminating adjacent wells, in order to fully disperse the sample into its diluent.
2.8.1.4.1.4. Dispense 50µl assay diluent (DILAS) into all the controls/calibrator and sample

wells. Check the colour of samples has turned to dark blue.

2.8.1.4.1.5. Incubate the microplate for 45 minutes at +37⁰C.

Important note: Strips have to be sealed with the adhesive sealing foil, supplied, only when

the test is carried out manually. Do not cover strips when using ELISA automatic instrument.

2.8.1.4.1.6. Wash the microplate with an automatic washer by delivering and aspirating 350µl

/well of diluted washing solution.

2.8.1.4.1.7. Pipette 100µl Enzyme Conjugate into each well, except the 1 st blanking well and

cover with the sealer. Check that this pink/red coloured component has been dispensed in all

the well except A1.

Important note: Be careful not to touch the plastic inner surface of the well with the tip

filled with the enzyme conjugate. Contamination might occur.

2.8.1.4.1.8. Incubate the microplate for 45 minutes at +37⁰C

2.8.1.4.1.9. Wash microwells as in step 6

2.8.1.4.1.10. Pipette 100µl chromogen/substrate mixture into each well, the blank well

included. Then incubate the microplate at room temperature (18-24 ⁰C) for 15 minutes.

Important note: Do not expose to strong direct illumination. High background might be

generated.

2.8.1.4.1.11. Pipette 100µl sulphuric acid into all the wells using the same pipetting sequence

as in step 10 to stop the enzymatic reaction. Addition of acid will turn the positive control and

positive samples from blue to yellow/ brown.

2.8.1.4.1.12. Measure the colour intensity of the solution in each well, as described at 450nm

filter (reading) and at 620-630 nm (background subtraction), blanking the instrument on

A1(mandatory)
2.8.1.4.2 Calculation of the cut-off

Negative control+0.350=cut-off

2.8.1.4.3 Interpretation of results

2.8.1.4.3.1 Sample OD/Cut-off=less than 0.9(mean that the result is negative)

2.8.1.4.3.2 Sample OD/Cut-off=0.9-1.1(mean that the result is equivocal)

2.8.1.4.3.3 Sample OD/Cut-off=Greater than 1.1(means than the result is positive)

2.8.1.5 Hepatitis B surface antigen (HBsAg) test

Detection of HBsAg in the serum/plasma indicates infection caused by the hepatitis B virus.

It is the first marker. It may be observed for 2-3 weeks before clinical and biological

symptoms. When it persists beyond six months it connotes chronic hepatitis.

2.8.1.5.1 Principle of HBs Ag test

The assay is a one-step enzyme immunoassay technique based on the principle of the

‘sandwich’ type using monoclonal antibodies and polyclonal antibodies for the detection of

the surface antigen on the addition of the red colour conjugate, colour of the wells becomes

red. After one and half hour incubation, the unbounded conjugate is removed by washing.

After substrate has been added the presence of the conjugate is shown by a change of colour.

2.8.1.5.2 Quality control

All controls are included in each run (1 positive &3 negative control).

2.8.1.5.3 Procedure of HBsAg

2.8.1.5.3.1. Carefully establish the sample distribution and identification plan.

2.8.1.5.3.2. Prepare the diluted washing solution.

2.8.1.5.3.3. Prepare the conjugate R6+R7 working solution.

2.8.1.5.3.4. Take out from the protective packing the support frame and necessary number of

strips (R1)

2.8.1.5.3.5. Distribute in the wells in the following order:

2.8.1.5.3.5.1. 100µl of negative control in wells A1, B1, C1, D1.

2.8.1.5.3.5.2. 100µl of positive control (R4) in well E1.

2.8.1.5.3.5.3. 100µl of the first unknown sample in well F1 if the well is not used as a control

well for the validation of the sample and conjugate deposition.

2.8.1.5.3.5.4. 100µl of unknown samples in well G1, H1, etc

2.8.1.5.3.6. Quickly dispense 50µl of conjugate solution (R6+R7) into all wells; the conjugate

solution must be shaken before use. Homogenize the reaction mixture.

2.8.1.5.3.7. When possible, cover the plate with new adhesive film.

2.8.1.5.3.8. Incubate the microplate for 1 hour 30 minutes (± 5 minutes) at 37⁰C ±1⁰C.

2.8.1.5.3.9. If necessary, remove the adhesive film. Aspirate the contents of add a minimum

of 370 µl of washing solution into each well. Aspirate again and repeat the washing a

minimum of 4 times. The residue volume must be lower than 10µl.

2.8.1.5.3.10. Prepare development solution (reagentR8+R9).

2.8.1.5.3.11. Quickly dispense into each well 100 microliter of prepared development

solution (R8+R9), freshly prepared before use. Allow the reaction develop in the dark for 30

minutes ± 5 minutes at room temperature (18-30⁰C). Do not use adhesive film during

incubation.
2.8.1.5.3 12. Add 100 microliter stopping solution(R10) by using the same sequence and rate

of distribution as for the substrate solution. Homogenize the reaction mixture.

2.8.1.6 HIV Ag-Ab

2.8.1.6.1. Carefully establish the sample distribution and identification plan
2.8.1.6.2. Prepare the diluted washing solution R2 and the conjugate 2 working
solution(R7a+R7b).
2.8.1.6.3. Take out from the protective packing the support frame and the necessary number
of strips(R1). Put the unused strips back in their packing. Close the packing and replace it at
+2-8⁰C.
2.8.1.6.4. Distribute in the well in the following order without prior washing of the plate:
2.8.1.6.4.1. 25 µl of conjugate 1(R6) in each well.
2.8.1.6.4.2. 75 µl of HIV Ag positive control (R5) in well A1
2.8.1.6.4.3. 75 µl of HIV Ab positive control (R4) in well B1.
2.8.1.6.4.4. 75 µl of negative control (R3) in well C1, D1, E1
2.8.1.6.4.5. 75 µl of specimen in well F1
2.8.1.6.4.6. 75 µl of specimen 2 in well G1 etc.
Homogenize the mixture by a minimum of 3 aspirations with 75µl or with a micropipette
shaker for 5 seconds. The sample distribution must begin immediately after the conjugate
negative and positive controls after the samples that are to be tested. After samples
distribution, the well containing conjugate 1 turn yellow-green to blue. It is possible to verify
the presence of the (sample + conjugate 1) in the wells.
2.8.1.6.5. When possible, cover the microplate with adhesive film. Press firmly all over the
plate to ensure a tight seal.
2.8.1.6.6. Incubate the microplate in a thermostat-controlled water-bath or microplate
incubator at 37⁰C ±1⁰C for 1 hour ±4 minutes.
2.8.1.6.7. If necessary, remove the adhesive film. Aspirate the contents of all wells into a
container for biohazardous waste. Add into each well a minimum of 0.370ml of washing
solution. Allow a soak time or at least 30 seconds. Aspirate again. Repeat this procedure a
minimum of two times (total minimum of three washes).
2.8.1.6.8. Quickly dispense 100µl of conjugate (R7a+R7b) into each well within the plate.
The conjugate must be shaken gently before use. Cover, if it is possible, with a new adhesive
film and incubate for 30 minutes (±4 minutes) at room temperature.
2.8.1.6.9. If necessary, remove the adhesive film, empty all the wells by aspiration and wash a
minimum of 5 times.
2.8.1.6.10. Prepare the enzymatic development solution(R8+R9).
2.8.1.6.11. Quickly dispense 80µl of prepared enzymatic development solution(R8+R9) in all
the wells. Allow the reaction to develop in the dark for 30 minutes ± 4 minutes at room
temperature. Do not use adhesive film during this incubation
2.8.1.6.12. Add 100microliter of the stopping solution (R10) using the same sequence and the
rate of distribution as development solution.
2.8.1.6.13. Carefully wipe each bottom. Wait at least 4 minutes after stopping solution
addition and within 30 minutes of stopping the reaction.
2.8.1.6.14. Read the optical density at 450/620-700 nm using a plate reader.
The substrate colour pink (negative samples) or blue (for positive samples) fades from the
wells which becomes colourless (for negative samples) or yellow (for positive samples) after
adding stopping solution
2.8.1.6.15 Check for agreement between spectrophotometer and visual reading and against
the plate and sample distribution and identification.
2.9 CENTER FOR INFECTIOUS DISEASE RESEARCH(CIDR)
CIDR is among the three centres in Microbiology Department where basic, translational and
clinical research were performed. Our highly interdisciplinary research focuses on the
transmission, evolution, immunology and control of infectious diseases among human
populations and environment, using cutting -edge tools in epidemiological and genetic
analysis, backed by focused field and experimental research. We collaborate with relevant
stake holders in microbial genomics research of pathogens, hosts, vectors and environment in
line with “One health” concept to monitor and control public health communicable diseases’
threats, nationally and internationally. Our scope spans a wide range of microbial research on
diseases with three research groups; Bacteriology, Parasitology and Immunology.
2.9.1 Bacteriology group
The bacteriology group conducts research on bacteriological infections of public health
importance which focus on;
2.9.1.1 Diarrhoea agents, respiratory tract infections, sexually transmitted diseases (STDs).
2.9.1.2 Microbial analysis of water, food and other environmental samples.
2.9.1.3 Antimicrobial resistance.
2.9.1.4 Opportunistic microbial diseases and zoonotic/nosocomial infections.
2.9.1.5 Capacity development on antibiotics stewardship for doctors and other healthcare
workers.
2.9.1.6 Operation on microbial repositories for research and academic purposes

2.9.2 Parasitology group

The parasitology group focuses on research on infections with parasitic origin with focus on;
2.9.2.1 Malaria and neglected tropical parasitic infections.
2.9.2.2 Genomics surveillance of antimalarial drug resistance markers.
2.9.2.3 Field evaluation of diagnostic devices for parasitic infections in collaboration with
relevant organization.
2.9.2.4 Implementation and applied research to improve diagnosis, promote rational (safety
and effective) use of diagnostic devices to improve health outcomes.

2.9.3 Immunology Group: conducts research on immunological marker of diseases of

public health importance.

2.10 Microbiological Culture

A microbiological culture is a method /technique of multiplying microbial organisms by
letting them reproduce in predetermined culture medium under controlled laboratory
conditions. Microbial cultures are used to determine the type of organism, its abundance in
the sample being tested, or both. It is one of the primary diagnostic methods of microbiology
and used as a tool to determine the cause of infectious disease by letting the agent multiply in
a predetermined medium.
2.10.1 Methods of Culturing Microorganisms
2.10.1.1 Streak Plate Method
A streak plate is the method of choice for obtaining pure culture. The streak plate technique is
used to isolate the organisms (mostly bacteria) from a mixed population into a pure culture.
The inoculum is streaked over the agar surface to “thin out” the bacteria. Some individual
bacterial cells are separated and well-spaced from each other. By streaking, a dilution
gradient is established across the surface of the agar plate. Because of this, confluent growth
occurs on the part of the plate where the bacterial cells are not sufficiently separated; in other
regions where few bacteria are deposited, separate macroscopic colonies develop. Each well-
isolated colony is assumed to arise from a single bacterium and represent a clone of a pure
culture.
2.10.1.1.1 Principle of Streak Plate Method
The inoculum is diluted by streaking it across the surface of the agar plate. While streaking in
successive areas of the plate, the inoculum is diluted to the point where only one bacterial cell
is deposited every few millimetres on the surface of the agar plate. An isolated colony is
formed when these lone bacterial cells divide and give rise to thousands and thousands of
new bacterial cells. Pure cultures can be obtained by picking well-isolated colonies and re-
streaking these on fresh agar plates.
2.10.1.1.2 Materials Required
2.10.1.1.2.1 A source of bacteria (stock culture, previously streaked agar plate, or any other
inoculum)
2.10.1.1.2.2 Inoculation loop
2.10.1.1.2.3 A striker/lighter
2.10.1.1.2.4 Bunsen burner
2.10.1.1.2.5 Lysol (10%v/v)
2.10.1.1.2.6 Agar plate (nutrient agar or any other agar medium.)

2.10.1.1.3 Procedure for Spread Plate

2.10.1.1.3.1 Use the loop gently.
2.10.1.1.3.2 Don’t make holes in the culturing medium.
2.10.1.1.3.3 Make the loop red hot, let it cool for 5 seconds, and then touch the culturing
medium briefly in a clean area to make sure it is cool.
2.10.1.1.3.4 Turn the dish 90 degrees while keeping the lid closed. Streak Area 2 with several
back-and-forth strokes, hitting the first streak a few times.
2.10.1.1.3.5 Make the loop red hot again. Turn the dish and streak through Area 3, making
sure to hit the last area more than once.
2.10.1.1.3.6 Burn the loop, let it cool, and turn the dish another 90 degrees.
2.10.1.1.3.7 Streak Area 4, get in touch with Area 3 more than once and pull out the culture as
shown.
2.10.1.1.3.8 Make the loop red hot before putting it down.

2.10.1.2 Spread Plate Method

The spread plate method is a microbiological laboratory technique for isolating and counting
the viable microorganisms present in a liquid sample by spreading a certain volume of the
sample over an appropriate solidified culture media. After the incubation at optimal
temperature, there will be the formation of evenly distributed discrete colonies all over the
surface of the culture media.

2.10.1.2.1 Procedure
2.10.1.2.1.1 Put the bent glass rod into a beaker and add enough 95% ethyl alcohol to cover
the bent part at the bottom.
2.10.1.2.1.2 Put a labelled nutrient agar plate on the turntable. Using a sterile pipette, put one
drop of sterile water in the middle of the plate. Then, put one loopful of sterile bacteria (e.g.
Micrococcus luteus) on top of the water drop. Mix slowly with the loop, and then put the lid
back on.
2.10.1.2.1.3 Take the glass rod out of the beaker and pass it through the flame of the Bunsen
burner so that the burning alcohol doesn’t run down your arm. The bent end of the rod should
point down. Let all of the alcohol burn off of the rod. Give the rod 10 to 15 seconds to cool
down.
2.10.1.2.1.4 Take off the lid of the Petri dish and turn the turntable.
2.10.1.2.1.5 While the turntable is spinning, move the sterile bent rod back and forth over the
surface of the agar. This will spread the culture across the surface of the agar.
2.10.1.2.1.6 When the turntable stops, put the cover back on. Soak the rod in alcohol and
burn it again on the burner.
2.10.1.2.1.7 Provided you don’t have a turntable, turn the Petri dish by hand and use the clean
bent glass rod to spread the culture.

2.10.1.3 Pour Plate Method

This method for separating one type of bacteria from another is to mix one loopful of
organisms with three tubes of molten nutrient agar in such a way that one of the plates will
have the right number of organisms for good isolation.
2.10.1.3.1 Procedure
2.10.1.3.1.1 Make the dilutions of the sample (which contain bacteria) according to sample
protocol and label the dilutions of each tube.
2.10.1.3.1.2 Label the Petri dishes according to respective dilutions.
2.10.1.3.1.3 Inoculate sample dilutions through a pipette in the centre of the Petri dishes.
2.10.1.3.1.4 Add molten nutrient agar to each Petri dish.
2.10.1.3.1.5 Swirl the Petri dishes for mixing and let them solidify.
2.10.1.3.1.6 After solidification, invert the microscope and incubate at 37⁰ C for 24 hours

2.11 Gram Staining

The Gram staining is a differential staining procedure used to divide bacterial cells into two
major groups known as Gram-positive and Gram-negative bacteria, which is used for the
classification and differentiation of microorganisms. Simple staining is used to observe the
shapes and arrangements of the bacterial cells. Gram staining not only helps the shapes and
arrangements, but also the thickness of the peptidoglycan cell wall of bacteria.
2.11.1 Materials used
Inoculating loop, Bunsen burner, glass slides, crystal violet, iodine, decolourizer, safranin
2.11.2 Procedure
2.11.2.1 The smear was prepared using a sterile glass slide.
2.11.2.2 An inoculating loop was used to pick a drop of water which was dropped on the
slide.
2.11.2.3 The inoculating loop was flamed and allow to cool and it was used to pick a distinct
colony which was emulsified on the glass slide.
2.11.2.4 The smear was then passed through the flame to heat fixed.
2.11.2.5 The smear was first stained with crystal violet a primary stain and left for 60 seconds
and it was flooded slowly with distilled water.
2.11.2.6 The smear was then stained with an iodine a mordant and left for 60 seconds and it
was flooded slowly with distilled water.
2.11.2.7 The smear was decolorized with ethanol and it was flooded immediately with
distilled water.
2.11.2.8 The smear was counter stained with safranin and left for 60 seconds and it was
flooded slowly distilled water.
2.11.2.9 The glass slide was allowed to air dry and was examined under oil immersion and
microscope.
2.11.3 Results
Gram-positive bacteria: blue/purple
Gram-negative bacteria: pink/red

2.12 Biochemical Tests

Biochemical tests are laboratory procedures that use specific chemical reactions to identify
and characterize microorganisms, such as bacteria. These tests are often used to identify the
presence of specific enzymes or metabolic pathways in a microorganism, which can help to
distinguish it from other microorganisms. There are several biochemical tests among them are
Kliglers Iron Agar (KIA) test, Motility Indole Urease test (MIU), Citrate test
2.12.1 Kliglers Iron Agar (KIA) test
This is a test used to differentiate bacteria based on their ability to ferment lactose, glucose,
produce hydrogen sulphide, gas production.
2.12.1.1 Lactose fermentation: Bactria that ferment lactose produces more acid and
intensifying yellow colour. When the colour occurs, it shows that the test is positive for
lactose fermentation. This is formed on the slant area of the solidified agar in a tube.
2.12.1.2 Glucose fermentation: Bacteria that ferment glucose produce acid also, this is also
positive by a colour change to yellow.
2.12.1.3 Hydrogen sulphide: Bacteria that produce hydrogen sulphide reduce ferric sulphide
to ferrous sulphide. The ferrous sulphide reacts the sulphur forming a black precipitate
(Ferrous sulphide). It is positive when there is a colour change from orange to black
2.12.1.4 Gas production: This is presence due to the presence of the gas bubbles in the
Durham tube placed inside the tube.
2.12.2 Motility Indole Urease (MIU) test
These consists of three tests which are motility, indole and urease test.
2.12.2.1 Motility test: This is show if the microorganism is motile. This is positive if there is
movement from the line of inoculum.
2.12.2.2 Indole test: This is used to detect the presence of tryptophanase enzyme in the
bacteria which hydrolyse tryptophan to indole, pyruvate and ammonia. This is the positive
when there is a formation of a red ring after 10 seconds of the addition of the Kovas reagent.
2.12.2.3 Urease test: This is used to detect the presence of the urease enzyme. This is positive
when there is a colour change from amber to pink.
2.12.3 Citrate test
This is used to detect the presence of bacteria that utilize citrate as their carbon source
All these tests were carried out in a sterile environment.
2.13 Antibiotic Sensitivity Testing (AST)
AST is the measurement of the susceptibility of bacteria to antibiotics. It involves measuring
the diameter of areas without bacterial growth, called zones of inhibition, around paper discs
containing antibiotics on agar culture dishes that have been evenly inoculated with bacteria. It
is also used to check for antimicrobial resistance of the bacteria. Examples of Antibiotics
used are: Erythromycin, Ampicillin, Amikacin, Ceftazidime, Clindamycin, Penicillin,
Vancomycin, Rifampicin, Imipenem etc.
2.13.1 Procedure of antimicrobial sensitivity testing.
2.13.1.1 The sample was collected which was then standardized using McFarland standard
and then streaked on the surface on an already prepared solidify agar then incubated at 37⁰C
for 18-24 hours.
2.13.1.2 After growth as been observed a distinct colony was taken suing a sterile inoculating
loop.
2.13.1.3 Which was then streaked on the surface on the solidified agar to obtain a pure culture
which was also incubated at the optimal temperature.
2.13.1.4 Then the antibiotics was then aseptically based on the surface of this pure culture
and the incubated at the optimal conditions.
2.13.1.5 Then the zone of inhibition was observed and it was measured and compared to the
standard to show susceptibility, resistance and intermediate.
2.14 Centre for Tuberculosis Research (CTBR)
The Centre for Tuberculosis Research (CTBR), is a unique collaborative Research Centre
founded in July 2017 in the Department of Microbiology of the Nigerian Institute of Medical
Research. The first National Tuberculosis Reference Laboratory in Nigeria is domiciled
within this Centre. This Centre contributes to the World Health Organization (WHO) End
Tuberculosis (TB) Strategy program through research, service and training. CTBR conducts a
range of multi-disciplinary research and training by dedicated scientists and support staff in
collaboration with universities, public and private sector organizations within the sub-region
and internationally.

2.14.1 Research
CTBR’s research is focused on tuberculosis (TB) epidemiology, drug resistance, evaluation
of novel diagnostics, genomics and laboratory management of Multi Drug Resistance (MDR)
TB patients. Current research is supported by the Nigerian Government (Nigerian Institute of
Medical Research and International organization (West African Node of Excellence for TB,
AIDS and Malaria [WANETAM]. CTBR also access grants for research activities.
3.0 Conclusion
3.1 Experience gained.
During my industrial training at Nigeria Institute of Medical Research I was able:
3.1.1 Interact with different people from different institution which helped to boost my
confidence.
3.1.2 I was able to experience how a laboratory works how real-life problems were
addressed.
3.1.3 I was able to see people with life threatening disease which make the infection look
more real to me
3.1.4 How was able to observe how important it is to maintain a sterile environment and
observe the laboratory rules.
3.1.5 I was able to observe how microbiological test were carried out.
3.1.6 I was able to operate microbiological equipment by myself.
3.1.7 I was able to help in administration of my team which also boost my confidence show a
good image of the institution.
3.1.8 I observed that student dressing, mode of speech is linked to the institution the student
came from which made me conclude that student must dress well in order to
3.2 Challenges
The organisation did not pay me salary during the period of my training, the cost of
transportation was also high.

3.3 Recommendations
3.3.1 I recommend that student should make more research and learn more virus due to its
great adverse effects in the environment.
3.3.2 I recommend that schools should give student referral to different organisation they can
work.
3.3.3 I recommend that more time should be given for the industry training so the student can
have more experience.

3.4 Conclusion
The aim of Students Industrial Work Experience Scheme (SIWES) gives students the
opportunity to apply knowledge gained in the university to real work environment, exposing
students to practical work methods not taught in the university. The aim of SIWES which was
to expose student’s machines and equipment, professional work methods and ways of
safeguarding the work areas and work in industries, laboratories, hospitals and other
organizations was achieved.