STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

TECHNICAL REPORT

AT

THE NIGERIAN INSTITUTE OF MEDICAL RESEARCH (NIMR).

6, EDMUND CRESCENT, YABA, LAGOS.

BY

POPOOLA MATTHEW IYIOLA

158859112

DEPARTMENT OF BIOCHEMISTRY

EKITI STATE UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE

DEGREE OF BACHELOR OF SCIENCE

AUGUST 15, 2018 – JANUARY 26, 2019

ACKNOWLEDGEMENT
First I would like to thank God almighty, for his grace in my life.

I am grateful to my family whose loves are with me in whatever I pursue.

This work would not have been possible without the ITF, CILPU and all concerned stakeholders
responsible for the Students’ Industrial Work Experience Scheme. I am especially grateful to
Nigerian Institute of Medical research for giving me the opportunity to participate in industrial
training under their esteemed research institute.

For me it was a unique experience to apply my theoretical knowledge of biochemistry and

science as a whole in practical solutions for problems encountered in medicine and the society at
large. It also helped in renewing my interest in biochemistry as a course.

I would like to express my sincere gratitude to the director general of the Nigerian institute for
medical research (NIMR) PROFESSOR BABATUNDE LAWAL SALAKO and the head of
department of the biochemistry division DR. OLUWAGBENGA AINA for giving me the
opportunity to carry out my internship within the organization.

I also would like to thank all the staff in the biochemistry division with their patience and
openness, they created an enjoyable working environment especially my industry based
supervisor DR. OLUSOLA AJIBAYE who has provided me extensive personal and professional
guidance and taught me a great deal about both scientific research and life in general.

Furthermore, I want to thank all the scientist and students, with whom I did my internship. We
experienced great things together and they have shown me the beauty in teamwork.

At last I would like to thank the department of biochemistry, EKITI STATE UNIVERSITY for
giving us a strong foundation on which more biochemical knowledge was easily built on.

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Contents
ACKNOWLEDGEMENT................................................................................................................................. 2

SUMMARY....................................................................................................................................................... 4

CHAPTER 1...................................................................................................................................................... 4

1.1 SIWES............................................................................................................................................................5
1.2 THE NIGERIAN INSTITUTE OF MEDICAL RESEARCH, (NIMR).................................................6
1.2.2 DIVISIONS AND UNITS......................................................................................................................7
1.2.3 NIMR ORGANOGRAM.....................................................................................................................14
1.2.4 RESEARCH GROUPS........................................................................................................................15
1.3.2 PRODUCTS AND SERVICES OFFERED BY NIMR......................................................................15
INTERNSHIP TRAINING ACTIVITIES.........................................................................................................17
2.1 MALARIA RESEARCH.........................................................................................................................17
2.1.1 MALARIA DIAGNOSIS.....................................................................................................................18
2.1.2 PRESERVATION OF MALARIAL PARASITE...............................................................................36
2.1.3ANTIPLASMODIAL STUDIES.................................................................................................................38
2.1.4 ANIMAL CARE...................................................................................................................................39
2.2 HEMATOLOGY UNIT...........................................................................................................................40
2.2.1 BLOOD GROUPING............................................................................................................................40
2.3 NUTRITION.............................................................................................................................................44
2.3.1 PROXIMATE ANALYSIS...................................................................................................................44
2.4 ISOLATION OF PHYTOCHEMICALS...............................................................................................49
2.4.1 QUANTIFICATION OF PHYTOCHEMICALS USING SOXHLET EXTRACTOR......................49
3.1 CHALLENGES ENCOUNTERED DURING THE SIWES PROGRAM...........................................58
3.2 EXPERIENCE GAINED.........................................................................................................................58
3.3 RECOMMENDATIONS...............................................................................................................................59
4.1 CONCLUSION.............................................................................................................................................. 60

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SUMMARY
The students industrial work-experience scheme (SIWES) instituted by the
industrial training fund (ITF) aims at developing the skills of Nigerian students to
contribute to the development of the economic and technological sector. This
executive summary gives an overview of my three months’ internship which
includes the activities, meetings and experiences obtained.
Activities performed in the biochemistry division of the Nigerian institute of
medical research (NIMR) included the fat constants tests, which checks for
adulteration of fat and oil, soxhlet extraction as a means of separating desired
compound from a source material, the use of Histidine-rich protein 2(HRP2) as a
rapid diagnostic test (RDT) for the diagnoses of malaria, packed cell volume
(PCV) analysis for the determination of the blood cell count, study of the
development of the malaria parasite in a mouse as well as the use of serial
passaging and freezing as a means of preserving the parasitic sample, application
of the thin and thick smear of plasmodium infected blood on microscopic slides
and staining using the giemsa stain for viewing under a microscope.
Attendance of the annual scientific conference in NIMR also created awareness to
the medical challenges faced in this country as well as giving an insight into the
scientific community in Nigeria.
It has been a wonderful experience in NIMR, as the organization creates a
conducive atmosphere where personal attributes such as critical thinking,
creativity, initiative, resourcefulness, time management, presentation skills,
interpersonal skills and so much more are developed and enhanced.

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CHAPTER 1
1.1 SIWES.
 HISTORY OF SIWES
The Student Industrial Work Experience Scheme (SIWES) is a skill training programme
designed to prepare and expose students of the university to the industrial work situation they are
likely to meet after graduation. The need for establishment of the scheme aroused when there
was a growing concern among industrialists that graduates of institutions of higher learning
lacked adequate practical background required for employment in industries.
In Nigeria, the Industrial Training Fund (ITF) established SIWES in 1973 to address among
other issues, the lack of relevant production skills of the Science, Engineering and Technology
graduates, needed by industry. The scheme was established by the Industrial Training Fund’s
Policy Document No.1 of 1973.
In line with the foregoing, SIWES programme was designed to complement classroom teaching
in the course of studies and to acquaint students with the skills needed in the industries after
graduation.

 BACKGROUND
The Student Industrial Work Experience Scheme (SIWES) started in 1974 with 748
students from 11 institutions of higher learning. By 1978, the scope of participation in the
scheme had increased to about 5, 000 students from 32 institutions. The Industrial Training Fund
(ITF), however, withdrew from the management of the scheme in 1979 owing to problems of
organizational logistics and the increased financial burden associated with the rapid expansion of
SIWES.
Following the resumption of management of SIWES by ITF in 1984, the scheme has
witnessed rapid expansion. Between 1985 and 1995, the numbers of institutions and students
participating in SIWES rose to 141 and 57, 433 respectively and the number has been increasing
dramatically till date.
Presently, participation in the scheme is limited to Science, Engineering and Technology
programmes in Universities and Polytechnics while in the Colleges of Education, NCE
programmes in Technical Education, Agriculture, Business, Creative Arts and Design, Computer
Studies and Home Economics are eligible.

 OBJECTIVES.
The Students’ Industrial Work-Experience Scheme (SIWES) is a planned and structured
programme based on stated and specific career objectives which are geared toward developing
the occupational competences of participants. The scheme became operational in 1974.
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The objectives include:
1. To provide an avenue for students in institutions of higher learning to acquire industrial skills
and experience during their courses of study;
2. To prepare students for industrial work situations that they are likely to meet after graduation;
3. To expose students to work methods and techniques in handling equipment and machinery
that may not be available in their institutions;
4. To make the transition from school to the world of work easier and enhance students’ contacts
for later job placements;
5. To provide students with the opportunities to apply their educational knowledge in real work
situations, thereby bridging the gap between theory and practice; and to enlist and strengthen
employers’ involvement in the entire educational process through SIWES

1.2 THE NIGERIAN INSTITUTE OF MEDICAL

RESEARCH, (NIMR).
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. Until the establishment
of National Institute for Pharmaceutical Research and Development (NIPRID) in Abuja, it was
the only institute in the country specifically dedicated for medical research. Mission and Vision

The VISION of the NIMR is to be an institution of excellence in basic, applied and operational
research for the improvement of health standard in Nigeria.

With a MISSION 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

The mandate of the institute under the enabling act of 1977 includes:
 Communicable diseases of public health importance in the country.
 Non-communicable diseases prevalent in the country.
 Basic, applied and operational research for the prevention and control of diseases
endemic in the country in co-operation with the federal and state ministries of health.
 Develop human and infrastructural capacities for clinical and biomedical research in
collaboration with medical schools, universities and other health related institutions.
 Disseminate the result of health research in the country through training courses,
scientific publications, conferences, and workshops and other communication channels to

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 the federal and states ministries of health, relevant stakeholders in the public and private
sectors as well as the general public.

1.2.2 DIVISIONS AND UNITS.

NIMR is divided into three units and seven divisions.

The units include: the library, monitoring and evaluation and the NIMR consult.

The divisions include: biochemistry and nutrition, clinical sciences, clinical diagnostic
laboratory, public health, molecular biology and biotechnology, human virology laboratory, and
microbiology.

Some of the divisions have sub-units, which aids in the effectiveness of works carried out.

1. BIOCHEMISTRY AND NUTRITION.

The Department conducts studies on the efficacy safety and cost-effectiveness of various
antimalarial agents for the treatment of malaria in the country. Other areas of research-focus of
the Department include the impact of nutrition on predominant infectious diseases in Nigeria. In
collaboration with other institutions, the Department is involved in studies to investigate the anti-
protozoa and anti-microbial properties of natural and synthetic organo-sulphur compounds. The
Department also is supported by the Global Fund, the Federal Ministry of Health and some
Pharmaceutical companies to conduct operational research support to the on-going roll back
malaria programme in the country.

The Department provides laboratory support for Internship, Doctoral and Post-Doctoral studies.
Department consists of the following units; Drug Analysis Unit, Nutrition Unit, Natural Products
Unit, Malaria Molecular Biology Unit, Operational Research Unit, Center for Research in
Traditional Complementary & Alternative Medicine (CRTCAM).

The department has been involved in the following research studies;

 Evaluation of ACT combinations with the view of advising appropriately what best
combinations to be adapted under the current WHO directives on ACTs for the Clinical
management of malaria in Nigeria.
 Molecular correlates of drugs-resistant Plasmodium falciparum in children in various
Nigerian communities.

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  Impact of various nutritional strategies in patients with HIV/AIDS, Tuberculosis and
Malaria.
 The roles of micro-nutrients and antioxidants have also been studied.

2 CLINICAL DIAGNOSTIC LABORATORY.

The unit provides efficient and quality diagnostic services for both staff and members of the
public at affordable costs. Supports research activities of the Institute as well as training of
students from universities and allied institutions from within the country; Collaborates with
researchers within and outside NIMR; Undertakes evaluation of kits, and mandatory food
handlers’ tests for companies and institutions.

3 CLINICAL SCIENCE.
The Clinical Sciences Division (CSD) conducts operational research as well as provides
comprehensive and world class care to thousands of Nigerians in the area of HIV, TB, child and
women’s health. The clinical services include the HIV Treatment and DOT Centers with support
from the AIDS Prevention Initiative Nigeria (APIN) and in collaboration with the National TB
Reference Laboratory at the Institute, respectively. CSD also provides health care services to
members of staff and their families.

The CSD is mandated to conduct research into human health problems in Nigeria, with particular
emphasis on;

 Communicable diseases prevalent in the country e.g. HIV/AIDS, TB

 Major non-communicable diseases prevalent in the country especially sickle cell disease,
Hypertension, Diabetes, Malnutrition, Malignancies etc.
 Maternal and Child health conditions
 Any other related matters as may be determined from time to time
 They also provide facilities for research in medicine in cooperation with medical schools,
universities and other institutions in and outside Nigeria
 Disseminate the results of medical research through training courses, publications etc.
 Social and laboratory-based research (including control interventions) on diseases such as
TB, malaria reproductive and sexual health, and HIV/AIDS as well as nutrition research

Some of these research activities are conducted in collaboration with local and international
collaborators such as APIN, Harvard PEPFAR and FHI360.
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4 MICROBIOLOGY.
The Microbiology division is made up of three units:

Center for Human Virology and Genomics: The unit conducts research into human virus, largely,
the HIV and the implications of the Highly Active Antiretroviral Therapy (HAART). HVL also
provides free screening and referrals for diseases of public health importance

Center for Tuberculosis Research and Diarrhea, Immunology & Parasitology Research Division

The divisions carry out researches in these areas.

5 MOLECULAR BIOLOGY AND BIOTECHNOLOGY.

The Department researches into the molecular epidemiology of diseases by parasites such as
Helicobacter pylori, Campylobacter jejuni, Salmonella typhi, EnterohemorrhagicEscherichia
coli (EHEC 0157:H7), and sexually transmitted infections including HIV/AIDS.

At the same time, some local foods have been bioengineered to control and prevent diarrhea
diseases, as well as improve their shelf life. The Department has also been involved in the local
production of antisera against Neisseria meningitides and ABO blood group. The Department
has also been screening local fruits and vegetables for antioxidant properties. While conducting
these researches, disease, status has been established and the appropriate local regimen effected.
Proper and accurate diagnosis of disease has been established.

The Department has also been in collaboration with various local and international bodies such
as Federal Ministry of Education, Health, FHI, Alexander von Humboldt (AvH), Germany,
INSERM, France, TWAS and Roche, Centre of Proteomics and Genomics (CPG), Cape-Town,
South-Africa, Universities at both National and International levels.

The Department is also involved in research activities of communicable and non-communicable

diseases. The Units in the Department comprise of;

 Molecular Epidemiology
 Biotechnology
 Hematology and Blood Disorders

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6 PUBLIC HEALTH AND EPIDEMOLOGY.
The Public Health and Epidemiology Department focuses on neglected tropical diseases,
communicable, non-communicable diseases, health system strengthening and other health
problems that are of public health importance in the country. The Department conducts
epidemiological (including control interventions), social and laboratory-based research on
diseases such as onchocerciasis, schistosomiasis, filariasis, malaria and, in addition nutrition,
reproductive and sexual health, and HIV/AIDS.

These research activities are conducted in collaboration with and support of local and
international bodies such as the States’ ministries of Health (e.g. Ondo, Ogun, Borno, Osun and,
Niger), National Control Programs (such as National Onchocerciasis Control Program, National
Schistosomiasis Control Program, Malaria Vector Control Unit/Roll Back Malaria), the African
Program on Onchocerciasis Control (APOC), the German Technical Cooperation (GTZ), the
office of Population Research, Princeton University, USA and the Swiss Tropical Institute.

Others include the Applied Research on Child Health (ARCH) project of Boston University
School of Public Health under the auspices of USAID in Washington, the WHO, UNICEF, the
National Institute for Communicable Diseases, the University of the Witwatersrand,
Johannesburg, South Africa and the Centre de RecherchéEntomologique du Benin, Cotonou.

7 ADMINSTRATIVE
The Administration Department provides the enabling environment for the realization of the
Institute’s research mandate through its Personnel Management functions. This involves routine
administrative discipline, interpretation and enforcement of the Public Service Rules (PSR),
provision of secretarial services to the Institute’s Management Committee, provision of legal
services, and issuance of cover for all assets, keeping of policy files and updating of staff
records. The Department also manages the Security Unit of the Institute.

8 INTERNAL AUDIT
The Internal Audit is responsible for routine and continuous internal control checks to ensure that
the Institute’s operations are conducted in accordance with government policies, rules and
regulations and other relevant circulars of government. The internal audit also ensures that the
institute obtains value for money in respect of payment for government goods and services. It
ensures goods or services procured follow due process, conform to specification without

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compromising quality. Internal Audit also carries out price verification on demand for goods
and services, and provides the management with strength for bargaining expenditure. The Unit
also ensures that government assets are accounted for, preserved, and maintained according to
standing regulations.

Responsibilities are as follows:

 Provide financial advice to the Management and other Employees

 Auditing of Financial Records/Documents of the Institute
 Carry out financial investigations Management based on government regulations
 Investigate on request financial matters and issues that are of relative importance and
concern to the management
 Conduct Prepayment audit of all transaction before any payment
 Ensure that paid vouchers were duly receipted acknowledged and accounted for
 Eliminate likely possible loopholes in payment for goods/services; and issuance of
certificate / endorsement for job done/goods supplied.
 Ensure that Institutional income and expenditures duly documented
 Ensured that projects/supplies conformed with official regulations & users specification
 Ensure that no fictitious payments are made

9 ACCOUNTS AND FINANCE

The Division is saddled with responsibility of handling financial matters of the Institute. The
major functions performed in this Division include the following:

 Maintenance of the Institute’s books of accounts and financial records

 Organizing and coordinating accounting activities of the Institute
 Preparing monthly, quarterly and annual operating statements; and
 Preparation of the Institute’s annual budgets
 Financial Grant Management

10 MONITORING & EVALUATION UNIT

The Monitoring and Evaluation Unit coordinates and supports Data management and Monitoring
and Evaluation activities of Departments, Units and Research groups in NIMR. These include
design of data collection tools, database design, data collection, data entry, provide M & E

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indicators for the management of projects and research activities and grant management.
Furthermore, we support these groups in the generation of their reports, analysis of data, use of
data generated to improve their activities and achieve their set objectives

Unit Activities includes:

Statistical support - The unit provides statistical consulting and collaborative support to the
researchers and research groups within and outside the institute.

Data and database management - They provide data management support to divisions and units
that have large dataset (HVL, CSD and DOTs Clinic) and Research groups; cleaning, updating
and organizing data generated from different departments and units; management of data
generated from field work for research groups; backup databases (on site and off site)

Grant management - The unit supports the Institute on:

 Grant management compliance

 Drafting, negotiating, interpreting and executing contracts with sub recipients and
consultants
 Evaluation of grant performance, regular technical reports to funders and liaising with
sub awardees
 Maintain NIMR’s SAM, Grants.
 .gov and eRA-Commons registration updates

11 LIBRARY AND INFORMATION CENTER

Library and information center at NIMR is a resource to literature and documentation mainly in
the field of malaria and other vector borne disease. This center uses LIBSYS software package
which consist of modules on acquisition, cataloguing, circulation, serial, OPAC, membership and
article indexing. All the collections in this library centre are electronically indexed.

12 WORK AND MAINTENANCE

The department is responsible to the Sec/Director of Administration and is saddled with the
responsibilities planning, developing and maintenance of all infrastructures, facilities, and
engineering services. Its function also includes the deployment of both human and material
resources to achieve cost effective development that will meet high engineering standards, liaises

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with the federal and state government’s agencies including the organized private sectors in
respect of approved developmental project.

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1.2.3 NIMR ORGANOGRAM

NIMR is a quango of the Federal Ministry of Health (FMOH). The current

Director-General/CEO is Professor Babatunde Lawal Salako, MBBS (Ibadan), FWACP, FRCP
(Edin), FRCP (Lond), MNIM and he answers directly to the Governing Board of the Institute.
The organization structure is well depicted in the organogram.

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1.2.4 RESEARCH GROUPS.
The Research Groups are intended to give a strategic direction to research activities in the
Institute so that research can be done better within timelines, targets and indicators aimed at
improving quality and productivity. The novel idea is also aimed at encouraging innovative
health research that will respond to national health priorities of the nation and contribute to the
attainment of health-related MDGs and vision 2020 (NIMR, 2002).

There are nine research groups in the Institute.

i. Malaria Research Group

ii. HIV/AIDS & TB Research Group
iii. Maternal, Reproductive and Child Health Research Group
iv. Non-Communicable Disease Research Group
v. Health System & Policy Research Group
vi. Neglected Tropical Diseases Research Group
vii. Clinical Trials Research Group
viii. Emergency and Preparedness and Response Research Group
ix. Immunology and Vaccinology Research Group
x.

1.3.2 PRODUCTS AND SERVICES OFFERED BY

NIMR.
Here is a highlight of the Institute’s products and services. However, the list is inexhaustible.

 The Institute conducts research into diseases of public health importance.

 Working in collaboration with the Federal and State Ministries of Health, the Institute
engages in basic, applied and operational research for the prevention and control of
endemic diseases.

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  The Institute serves as venue for several health-related programmes organized by other
organizations or industry.
 The Institute engages in community services:
a) Enlightenment campaign in schools and market places in order to reduce the
morbidity and mortality associated with common diseases,
b) Free screening for life-threatening diseases such as cervical cancer, diabetes,
hypertension and obesity.
 The Institute provides clinical services to the public at affordable price.
 The TB unit provides support services for various components of the National TB
Control Programme.
 NIMR provides free HIV Testing and Counseling and Treatment for HIV positive
patients.

1.2.10 STAFF’S STRENGTH.

The information below was provided by the HOD of the Administrative Division, Alhaji A.S
Yunusazazzau during the Institute’s 5th Annual International Scientific Conference held from
the 11th to 14thof November, 2018.

‫ךּ‬ The Institute has a total of 317 staff on its pay roll.
‫ךּ‬ This figure comprises 172 males and 145 females; 69 research fellows and 258 non-
research fellows (NIMR, 2002).

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CHAPTER 2.
INTERNSHIP TRAINING ACTIVITIES.
The training activities performed during this programme were based mainly on the malaria
research and nutrition.

2.1 MALARIA RESEARCH

The Biochemistry Department of NIMR specialized mostly and it is renowned for its edge in
malaria research. I was taken through an
experimental and theoretical lectures on
basic techniques in Malaria Research,
which were centered mainly on life-cycle
of the malaria parasite, preservation, and
it’s Diagnosis.

Malaria is a mosquito-borne infectious

disease affecting humans and other
animals caused by unicellular
microorganisms belonging to the general
Plasmodium. Malaria causes symptoms
that typically include fever, tiredness, Figure 1: A mosquito
vomiting, and headaches.

The malaria parasite lifecycle (CDC).

The natural history of malaria involves cyclical infection of humans and female Anopheles
mosquitoes. In humans, the parasites grow and multiply first in the liver cells and then in the red
cells of the blood. In the blood, successive broods of parasites grow inside the red cells and
destroy them, releasing daughter parasites (“merozoites”) that continue the cycle by invading
other red cells.

The blood stage parasites are those that cause the symptoms of malaria. When certain forms of
blood stage parasites (gametocytes, which occur in male and female forms) are ingested during

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blood feeding by a female Anopheles mosquito, they mate in the gut of the mosquito and begin a
cycle of growth and multiplication in the mosquito.

Figure 2: Lifecycle of malaria parasite

After 10-18 days, a form of the parasite called a sporozoite migrates to the mosquito’s salivary
glands. When the Anopheles mosquito takes a blood meal on another human, anticoagulant
saliva is injected together with the sporozoites, which migrate to the liver, thereby beginning a
new cycle. Thus the infected mosquito carries the disease from one human to another (acting as a
“vector”), while infected humans transmit the parasite to the mosquito, in contrast to the human
host, the mosquito vector does not suffer from the presence of the parasites.

2.1.1 MALARIA DIAGNOSIS

Amongst the different methods of malaria diagnosis, only two of them were carried out during
the course of my training. These includes; the microscopic method and the Rapid Diagnostic
Test (RDT).

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2.1.1.1 MICROSCOPIC EXAMINATION OF MALARIA
PARASITE
The mainstay of malaria diagnosis has been the microscopic examination of blood, utilizing
blood films. There are two types of blood films preparation test used in the malaria parasite test.
They are thick and thin blood films.

The accepted laboratory practice for the diagnosis of malaria is the preparation and microscopic
examination of blood films stained with Giemsa, Wright’s, or Field’s stain. Blood obtained by
pricking a finger or earlobe is the ideal sample because the density of developed trophozoites or
schizonts is greater in blood from this capillary-rich area. Blood obtained by venipuncture
collected in heparin or Sequestrine (EDTA) anticoagulant-coated tubes is acceptable if used
shortly after being drawn to prevent alteration in the morphology of white blood cells (WBC)
and malaria parasites. Both thick and thin blood films should be prepared.

Figure 3: Blood films

THICK BLOOD FILM.

The thick blood film concentrates the layers of red blood cells (RBC) on a small surface by a
factor of 20 to 30 and is stained as an unfixed preparation using Field’s stain or diluted Wright’s
or Giemsa stain. The thick blood film provides enhanced sensitivity of the blood film technique
and is much better than the thin film for detection of low levels of parasitaemia and reappearance

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of circulating parasites during infection recrudescence or relapse. The lysis of the RBC during
the staining process can make the process of scanning for parasites more difficult until
experience is gained in finding the parasites among the WBC and platelets.

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THIN BLOOD FILM.

The thin blood film is methanol fixed and stained with diluted Giemsa or Wright’s stain using
buffered water at pH 7.2 to emphasize the parasite inclusions in the RBC. Because of the fixed
monolayer of RBC available in this procedure, the morphological identification of the parasite to
the species level is much easier and provides greater specificity than the thick-film examination.
The thin blood film is often preferred for routine estimation of the parasitaemia because the
organisms are easier to see and count. The ability to count parasites in sequential blood films
enables the response to therapy to be monitored, particularly for P. falciparum infections.

Materials: Pasteur pipette, oil immersion, Clean grease- free microscopic slide, diluted Giemsa
stain pH of 7.1-7.2, small plastic bulb pipette, smooth edged slide spreader, absolute methanol
(methyl alcohol) or ethanol (ethyl alcohol), microscope, fresh blood sample (anticoagulated).

PROCEDURE FOR MAKING THE THICK FILM

 Wash hands (before and after the procedure) and wear a pair of new gloves.
 Thick film can be made on the same slide as thin film or separately.
 Put two to three drops of blood on the slide.
 Use the spreader to make a thick film on the slide. Make sure the blood is even and
allowed to dry. Then stain with working solution of Giemsa stain and allow for minutes
depending on the percentage stain prepared e.g. 30minutes for 3% stain preparation.
 Rinse off the stain by gently pouring water to wash off any sticky attachment and surface
and then stand on the drying rack to dry.
 Put a drop of oil immersion on the slide than start the microscopic examination using
100x objective lens of the compound microscope for plasmodium species.

EXAMINATION OF THICK BLOOD FILMS.

A recognized way of estimating the number of parasites present in 1 μl of blood is to use a

standard value for the WBC count (8,000 WBC/μl). Counting the number of parasites present
until 200 WBC have been seen and then multiplying the parasites counted by 40 will give the

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parasite count per microliter of blood. If an accurate WBC count is known, this can be used to
give a more accurate figure with appropriate adjustment of the multiplication factor.

Figure 4: A microscope

RESULT INTERPRETATION

Malaria parasite test is reported based on the degree of infection of malaria parasite.

 +1-10 parasite per 100 fields. Scanty malaria parasites seen.

 ++11-100 parasite per 100fields. Numerous malaria parasites seen.
 +++1-10 in every field. Very many malaria parasites seen.
 A slide is negative when no parasite is seen in 100 fields.

Parasite levels (or parasitaemia) can be detected as few as 5 parasites/µL blood.

Procedure for making the thin film

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  Get a clean grease-free microscopic slide. Then label slide appropriately with the
patient’s name.
 Put a small drop of the blood sample and slide toward the end of the slide on which the
smear is to be made.
 With your left hand, hold the edges of the slide that is away from the blood and with the
right hand; hold the spreader at an angle of 25-45 degree to the slide.
 Move it back to make contact with the drop of blood. The drop should spread out quickly
along the line of contact of the spreader with the slide by capillary action.
 The spreader is pushed smoothly and rapidly along the slid, drawing the blood behind it
until the smear is formed and allow the film to dry.
 Fix the film by placing it in absolute methyl alcohol (methanol) for 30 seconds.
 Then stain with Giemsa stain and allow for minutes depending on the percentage stain
prepared for visualization of the parasite in the RBC under the microscope(e.g. 30minutes
for 3% stain preparation) rinse and allow drying on the drying rack.
 Place a drop of oil immersion to the lower third of the film and spread the oil to cover
most of this part of the film and examine the film with 40x objective to 100x objective.
 Check the staining, morphology and distribution of the cells and to detect malaria
schizonts, gametocytes and trophozoites (the very small rings of Plasmodium falciparum
may not be seen with this objective). P. malariae and P. ovale parasites can often be
found on the edges of a thin film.

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Figure 5:How to make a thin film

Examination of thin blood films.

The parasitaemia may be estimated by examination of a well-stained thin blood film. This is
usually accomplished by noting the number of parasitized RBC (not individual parasites) seen in
10,000 RBC (equal to approximately 40 monolayer cell fields of a standard microscope using the
100× oil immersion objective; however, microscopists are advised to calculate the average
number of cells per microscope field of view for their own microscopes) and expressing the
number of parasitized cells seen as a percentage. The approximate numbers of parasites present
in 1 μL of blood can be calculated by assuming that 1 μL of blood contains 5 × 106 RBC;
therefore, a 1% parasitaemia will contain 1 parasite/100 RBC or 50,000 parasites/μl of blood.
Similarly, a 0.1% parasitaemia will contain 5,000 parasites/μL of blood. This may be corrected
to exact counts if the total RBC count per microliter is known.

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 GIEMSA STAINING
Giemsa's solution is a mixture of methylene blue, eosin, and Azure B. The stain is usually
prepared from commercially available Giemsa powder.

The preparation of Giemsa stock solution includes 3 reagents; Giemsa powder, Methanol and
Glycerol. 3.8g of Giemsa powder is mixed with 250ml of Methanol and Glycerol.

PREPARATION OF GIEMSA WORKING SOLUTION

These solutions are prepared by diluting the Giemsa stock solution with the appropriate volume
of water. The concentrations of Giemsa usually
prepared are 3% and 10% solutions.

3% concentration is used to stain a large number of

slides. It can be used for research purposes. 10%
concentration is used for routine purposes.

It is estimated that about 3ml of Giemsa working solution is required for the staining of a slide.
Since the stain is to be prepared fresh, it is important to consider the number of slides to be
stained.

Figure 6: Giemsa stained slides If there are N number of slides bearing the number of
slides to be stained, then the total volume of Giemsa stain working solution required is 3mL × N
= 3N mL.

Using 3% stain: 3/100 × 3N ml gives the volume of the Giemsa stock solution to be diluted. This
amount is measured out and diluted to the 3N ml by adding buffered water or distilled water.

2.1.1.2 MALARIA DIAGNOSIS USING RDTs

RDTs are lateral flow immuno-chromatographic antigen-detection tests, which rely on the
capture of dye-labeled antibodies to produce a visible band on a strip of nitro-cellulose, often
encased in plastic housing, referred to as cassettes. I.e. the test is based on migration and
separation of immune molecules in a wet (buffered) medium

Malaria RDTs is an alternative way of quickly establishing the diagnosis of malaria infection by
detecting specific antigens (proteins) produced by malaria parasites in the blood of infected

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individuals.The RDT tests are still regarded as complements to conventional microscopy. The
tests are simple and the procedure can be performed on the spot in field conditions.

Malaria RDTs detect specific parasite antigens in blood, mainly Histidine Rich Protein 2 (HRP2)
or Plasmodium lactate dehydrogenase (pLDH).

Plasmodium aldolase and Plasmodium Glutamate dehydrogenase precipitated by host antibodies

are other antigens that are used in some tests. Some RDTs can species specific (e.g. Malaria
RDTs pre-coated with monoclonal antibody HRP2 detects only P. falciparum) while others
detect one or more of the other three species of malaria parasites that infect humans.

HRP2 MALARIA TESTS KIT.

Figure 8: CareStart™Malaria RDTs kit Figure 7: Malaria

RDT cassette
The test kit consists of:

₰ Instruction manual
₰ Test device
₰ Alcohol swab
₰ Sample pipette
₰ Lancet
₰ Buffer solution.

PROCEDURE
₰ Put on protective wears

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 ₰ Label cassette with patient’s information/code.
₰ Wipe the area of the fingertip to be pricked with an alcohol swab for the purpose of
sterilization.
₰ Squeeze the area and prick with lancet.
₰ Discard the lancet immediately in the sharp box.
₰ Wipe out the first blood with sterile cotton wool.
₰ Collect about 5ul of blood using the sample pipette
₰ Add blood into the sample well, "S" on the test kit.
₰ Add 60ul of buffer solution or as specified by the kit manufacture into "A" well.
₰ The result is then read after 20 minutes or as specified by the kit manufacture.

Figure 9: How to use RDTs

INTERPRETATION OF RESULTS

POSITIVE: The presence of two colour bands on the C line and T line indicates a positive result
and presence of Plasmodium spp.

NEGATIVE: The presence of only one colour band on the C line indicates a negative result and
absence of Plasmodium spp.

INVALID: A result is invalid, if there is no colour band on the C line and the test must be
repeated.

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Figure 10: Interpretation of malaria RDTs results

LIMITATIONS OF THE RDTs:

The RDT may not be able to detect some infections with lower numbers of malaria parasites
circulating in the patient’s blood stream. Also, there is insufficient data available to determine
the ability of this test to detect the two less common species of malaria, P. oval and P. malariae
as most RDT kits are P. falciparum specific. Therefore, all negative RDTs must be followed by
microscopy to confirm the result. The PAN-Aldolase method of malaria diagnosis is efficient in
the diagnosis of all malaria parasites.

The kit may produce false negative test when the concentration of the malaria parasite per blood
titer is very high i.e. it has exceeded the RDT functional threshold.

And it may also report a positive test for an uninfected patient or after a successful treatment of
malaria. This is would be due to the presence of the HRP II protein still being in the blood, even
though the individual is cured of the parasite. The effect of this limitation can last up to 4 month
after the individual has being cured of the malaria parasite.

2.1.1.3 MOLECULAR DIAGNOSIS AND CHARACTERIZATION OF

MALARIA PARASITE
DNA EXTRACTION

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DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells. A number
of different methods are available for the isolation of DNA from whole blood, including salting
out/salt precipitation, phenol/chloroform extraction, silica gel extraction, proteinase K extraction
and anion exchange. The choice of method depends on many factors including the required
quantity, purity required for downstream application, time, molecular weight of DNA and
expense.

The general procedure for extracting nucleic acids consists of three consecutive steps:
disintegration of cells or tissues (cell lysis), inactivation of intracellular nucleases, and separation
of nucleic acids from the other cell components.

Once extracted and optimum purity attained, DNA can be used for molecular analyses including
PCR, electrophoresis, sequencing, fingerprinting and cloning.

DNA EXTRACTION FROM DRY BLOOD SPOTS/FILTER PAPER USING PHENOL-

CHLOROFORM ISOAMYL ALCOHOL METHOD

In this protocol the cell lysate is mixed with phenol, chloroform and isoamyl-alcohol for the
separation of nucleic acids from proteins. This method gives a high yield, but traces of organic
solvents often contaminate the sample.

DNA can be isolated from whole blood (EDTA) or a cell pellet following plasma separation
from an EDTA sample.

DNA should be processed as soon as is practicable but a specimen can be stored at 4°C for 48
hours prior to processing or alternatively can be stored directly at -80°C for DNA processing at a
later date

PRINCIPLE

This protocol allows purification of DNA by the addition of phenol and chloroform, leading to
the appearance of two phases: an upper aqueous phase containing the nucleic acids and an
organic phase containing proteins solubilized in phenol and lipids dissolved in chloroform.

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For purifying DNA, phenol should have a pH≈7-8. Subsequently the DNA is precipitated from
the aqueous phase with isopropanol or absolute ethanol and washed with 70% ethanol to remove
salts and small organic molecules that may still be present in the sample. Finally the DNA is re-
suspended in an appropriate buffer.

PROCEDURE

 Blood samples were applied in spots on filter paper.

 A punch was dipped into bleach, distilled water and ethanol.
 The punch was used to punch the blood spot from the filter paper into 1.5ml Eppendorf
tube.
 250µl of tail digestion buffer was added, incubated overnight at 40-450C.
 It was spun at 13,000rpm using a centrifuge.
 200µl aqueous phase was transferred to 1ml 100% ETOH to precipitate DNA.
 It was spun at 13,00rpm to pellet then the 100% ETOH was decanted into a sink.
 The pellet was dried for 10min on bench top.
 100µl TE buffer was added to re- suspend the DNA.
 It was stored at 40C until ready for use.

Figure 11: Eppendorf tubes

Figure 12: Micro-fuge

The yield indicates the value obtained from experimental data provided by the different biobanks
that use them.

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The quality of the obtained samples is determined by their purity, integrity and functionality.

Purity:

The purity of the sample is related to the value of maximum absorbance of the nucleic acids
detected at a wavelength of 260 nm.

The absorbance ratio A260/A280 reveals whether the DNA obtained is contaminated by the
presence of aromatic compounds, since they absorb at a wavelength of 280 nm. This ratio is very
stable and it is generally considered that the DNA is of optimal quality when the A260/A280
ratio is more than 1.8. An A260/A 280 ratio > 2.1 is indicative of a significant amount of RNA
present in the sample. Conversely, if this ratio is low (A260/A280 <1.6) the sample is
contaminated with proteins or phenols. In case of contamination with proteins it will be
necessary to carry out an additional treatment to remove the proteins from the DNA solution (for
example, addition of proteinase K). If contamination is due to the presence of phenols, the
sample needs to be cleaned with chloroform, isoamyl alcohol and ethanol.

The absorbance ratio A260/A230 is used as an additional measure to determine DNA purity, as
at 230 nm the maximum absorbance of salts, carbohydrates or other contaminants present in the
solution is detected. DNA is generally considered to be pure when the A260/A230 ratio is around
1.5-2.2. A ratio of less than 1.5 may be indicative of the presence of contaminants in the sample.
However, it must be kept in mind that the information provided by this measure is not as
accurate as the A260/A280 ratio, and that it can be
distorted by a low concentration of DNA in the sample
since one would be overestimating the concentration of
salts in the resuspension buffer.

Integrity:

Electrophoresis in a 0.7% (w/v) agarose gel allows assessing the integrity of the DNA sample. A
DNA sample of high integrity shows a single, perfectly defined band at the top of the agarose
gel. A degraded DNA sample will show a smear in the gel, which will be more pronounced when
the degradation of the sample is higher.

Functionality:

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The fact that a DNA polymerase enzyme can use a DNA sample as a template in a PCR reaction
indicates that the purity of the sample is optimal. Moreover, one can assess the degree of sample
integrity based on the size of the amplified fragment.

POLYMERASE CHAIN REACTION (PCR)

PCR is a molecular biology technique used to make copies of a specific DNA segment quickly
and accurately.

A single copy of a DNA sequence is exponentially amplified to generate thousands to millions of

more copies of that particular DNA segment.

PCR is a three-step process that is carried out in repeated cycles

The initial step is the denaturation, or separation, of the two strands of the molecule
accomplished by heating the purified DNA sample to 95OC.

In second step is the temperature is reduced to about 55OC so that the primers can anneal to the
template.

In the third step the temperature is raised to about 72OC, and the DNA polymerase begins
adding nucleotides onto the ends of the annealed primers. The cycle is further repeated 24 or 29
times more

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The whole Polymerase chain reaction is easily carried out using an automated machine called a
“Thermal cycler”

Figure 13: Polymerase chain reaction

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Figure 14: Thermal cycler

AGAROSE GEL ELECTROPHORESIS

The agarose gel electrophoresis is carried out to confirm DNA amplification.

PREPARATION OF 1% AGAROSE GEL

₰ Dissolve 1.0g of agarose in 100ml of electrophoresis buffer (TBE or TAE) by heating in

a microwave.
₰ After heating and fully dissolving the agarose add 10uL of SYBR SAFE/Ethidium
Bromide directly into the liquid via pipette, mix by gentle swirling.
₰ Use this mixture to cast a gel

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 ₰ Place the gel tray into the gel box so that the gasket (Ends with rubber strip) forms a seal
against the walls of the gel box make sure to press the gel tray all the way down so that
the gel box and gel tray are level.
₰ After the gel mix has cooled to about 60OC (Higher temps will damage and warp the gel
box), carefully pour the mix into the gel tray.
₰ Upon pouring the gel mix, remove bubbles or debris and immediately insert the gel comb
with the desired number of teeth/wells.
₰ Allow the gel to solidify completely. Then lift the gel tray out of the gel box turn it 90O
and replace it into the gel box with the comb closest to the cathode.
₰ Pour running buffer into the gel box to fill the chamber and completely submerge the gel
(300ml).
₰ Carefully remove the comb using a light tapping motion to avoid damage to the wells.

LOADING SAMPLES AND RUNNING GEL ELECTROPHORESIS

 Pipette 10uL of your sample into a clean tube.

 Add 2uL of loading dye (Bromophenol blue) (Blue dye) for visual tracking.

 Mix by low vortex and spin on mini-fuge to ensure all sample is at the bottom of the well.

 Plan out your gel before pipetting and create a reference chart/diagram to ensure correct
samples are put into the correct wells. Also that you have a negative and positive control
in addition to a hyper-ladder on one or both ends of the gel (First and last well).

 Carefully pipette all of sample into the correct well on your gel using 10xL pipette tips
(Or any compatible extended length tips)

 Add 10uL of the hyper-ladder for reference/comparison in at least one or if possible both
ends of your gel (First and last wells)

 Run the gel at 100V for 30-45mins to migrate gel loading dye front.

 Check to see that loaded materials has run towards the positive end of the gel box
sufficiently to visualize the band and identify the specific weights.

 Visualize the gel on a bio-imaging system and photography or UV Trans illuminator.

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Figure 15: visualization of DNA bands on Agarose gel using UV-illuminator

2.1.2 PRESERVATION OF MALARIAL PARASITE

Malaria Parasite (Plasmodium spp.), has been renowned for its virulence in patients if not
properly taken care of. This has triggered its importance in health research and development, and
therefore have presented the need for an efficient method for preservation of the parasite. In
recent years, several methods of preservation have been developed. During my internship
experience I was taken through the method of Serial Passaging and the Freezing Method.

 Passaging
 Freezing

2.1.2.1 PASSAGING
This is usually done by using a capillary tube, also called a microhaematocrit to obtain blood
from a mouse infected already with Plasmodium berghei (this species cannot affect humans)
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majorly via the ocular sinuses. About 0.1ml of blood is mixed with 3ml of Phosphate Buffer
Saline. The buffered parasitized blood is then administered into a fresh mouse or mice as the
case maybe via the intra-peritoneal route. A mouse without the parasite contains 5.0 ×108red
blood cells in 1mL of blood. When the parasite is introduced, the red blood cell is reduced to 3.0
×108.

To know the number of red blood

cells in 0.1ml of blood sample

1mL = 300million

0.1mL = x

x = 0.1 x 300,000,000

=30,000,000 RBC

Figure 16: Inoculation of mouse with malaria parasite via the intraperitoneal
route

2.1.2.2 FREEZING
A particular volume of blood sample is collected via a capillary tube from an infected animal
into an EDTA (Ethylene diamine tetra-acetic acid) –an anti-coagulant tube. It is then centrifuged
at 3500g for 10mins.The plasma containing clotting factor (fibrinogen) is seeded into the
supernatant after centrifugation by it interaction with EDTA. The supernatant is then collected
carefully using a Pasteur pipette leaving only the red blood cells containing the parasite. Equal
volume of Glycerolyte-57 is added to the red blood cell which is marked and dated and can be
stored at -80˚celcius (186K).

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2.1.3ANTIPLASMODIAL STUDIES
This involved the research for the development and screening of selected plant extracts usually
of plants used in traditional medicines, their toxicological evaluation and the therapeutic efficacy
as an anti-malaria drug using laboratory rodent models. The tests used in this study includes

2.1.3.1 SUPPRESSIVE TEST

This involved injecting donor mice with a chloroquine sensitive strain of Plasmodium berghei.
After about two hours of infection with parasites, the mice systematically assigned into treatment
groups are treated for four days with whole plant extracts or in combination with other
compounds administered orally. On the fifth day, blood is collected from donor mice by
venesection of the tail and a smear of thin and thick malaria blood films is made for examination
under the microscope.

The blood films were fixed using methanol, they were stained with 10% Giemsa at pH 7.2 for 10
minutes and parasitaemia examination was done using the microscope (× 100 magnifications).
Percentage suppression was then calculated.

Parasitaemia∈negative control−Parasitaemia∈study group

% Suppression=
Parasitaemia∈negative control

2.1.3.2 CURATIVE TEST/THERAPEUTIC EFFICACY

STUDY (TES)
Monitoring the efficacy of antimalarial medicines is a key component of malaria control. The
appearance of Plasmodium falciparum resistance to many antimalarial medicines is a concern
against malaria. Protecting the efficacy of artemisinin-based combination therapies (ACTs) as
the current first- and second-line treatment for Plasmodium falciparum is among the top global
public health priorities.

Antimalarial drug efficacy was assessed through therapeutic efficacy studies (TES). TES are
conducted in a controlled environment in which drug administration was supervised, the results
of microscopic examinations of blood films were validated, and the origin and quality of the
drugs are verified from the day of inoculation till the final day of the study usually for 10 – 28
days.

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Therapeutic outcomes were assessed on the final day of the study.

In NIMR, the curative antimalarial potential of plant extracts was done by employing the method
described by Ryley and Peters (1970). Donor mice were injected via the intraperitoneal route
with standard inoculum of Plasmodium berghei infected erythrocytes on the first day (day 0).
After 72 hours, and following confirmation of parasitaemia, the mice were grouped and treated
with prepared plant extracts at different concentrations, treatment with chloroquine served as a
positive control and an equal volume of distilled water as negative. Treatment lasted for 5 days at
single dose per day after which blood smears were collected and examined microscopically to
monitor the parasitaemia level.

2.1.3.3 CHEMOPROPHYLAXIS
Malaria chemoprophylaxis is the preventive treatment of malaria.

The evaluation of the prophylactic potential of selected plant extracts involves the administration
of whole plant extracts at different concentration into mice divided into groups against a positive
control group administered with chloroquine and negative control group administered with
distilled water. All drug administration were done via the intraperitoneal route and the
administration was continued for 5 days.

All treatment was initiated on day 0 and continued until day 4; the mice were all infected with
the malaria parasite. Blood smears were then made from each mouse 72 hours after infection.
Increase or decrease in parasitaemia was then determined.

2.1.4 ANIMAL CARE

Animal care is an essential of scientific studies, unless stated otherwise. This is to ensure
external environmental factors do not interfere with result experiments. Laboratory animals
especially mice are a major tool for malaria research unit. They are from time to time inoculated
with Plasmodium berghei for series of tests ranging from test to check the possible anti-
plasmodia activity of plant extracts to acute toxicity tests for food and drugs and also, the
preservation of the parasite. Laboratory animals were also used in cancer research.

Working with laboratory animals has made research works relatively quicker as compared to the
old times and as such the sine qua non for animal care. Animal care involved but was not limited
to:

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 Administration of food (rat chow) and drugs to the laboratory animals
Changing of beddings (wood-shavings) and water at regular intervals or when
necessitated
Handling of rats/mice in the most humane way possible to avoid abuse.
Differentiation and selection of animals based on characteristics such as gender and body
mass in preparedness for experimental study.
Inspection of the animal models and reporting any unusual observations to the supervisor
Collection of blood from mice by venesection of the tail and making of thin and thick
malaria blood films.

2.2 HEMATOLOGY UNIT

Hematology is the study of the morphology and physiology of blood in health and disease. It is
concerned with the diagnosis and monitoring of diseases of the blood and blood forming organs.
It includes problems with RBC, WBC, platelets, blood vessels, bone marrow, lymph nodes,
spleen, and proteins involved in bleeding and clotting (hemostasis and thrombosis).

Tests carried out in this unit include; packed cell volume (PCV), Erythrocyte Sedimentation Rate
(ESR), blood grouping, blood genotyping and differential counting.

During the course of my programme, I was only able to participate in blood grouping

2.2.1 BLOOD GROUPING

Blood grouping (also called blood typing) is a test that tells what specific type of blood an
individual have. The type of blood an individual have depends on whether or not there are
certain proteins, carbohydrates, glycoproteins, or glycolipids called antigens present on a
person’s red blood cells, depending on the blood group system. Some of these antigens are also
present on the surface of other types of cells of various tissues. Several of these red blood cell
surface antigens can stem from one allele (or an alternative version of a gene) and collectively
form a blood group system.

Blood are often grouped according to the ABO and the Rh blood group systems. This method
breaks blood down into four types namely; A, B, AB, and O with +, - or null denoting RhD
status.

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2.2.1.1 PRINCIPLE OF ABO BLOOD GROUP SYSTEM
The ABO blood group system involves two antigens and two antibodies found in human blood.
The two antigens are antigen A and antigen B. The two antibodies are antibody A and antibody
B. The antigens are present on the RBC and the antibodies in the serum. Regarding the antigen
property of the blood all human beings can be classified into 4 groups, those with antigen A
(group A), antigen B (group B), with both antigen A and B (group AB) and those with neither
antigen (group O). The antibodies present together with the antigens are found as follows:

i. Antigen A with antibody B

ii. Antigen B with antibody A
iii. Antigen AB has no antibodies
iv. Antigen nil (group O) with antibody A and B

When blood sample obtained from a patient is mixed with antibodies type A, B and O antigens
and the sample is checked for agglutination reaction. Agglutination can only if blood the blood
contains antigen similar to the antibody (Antigen A agglutinates the antibody A and Antigen B
agglutinates the antibody B). If blood cells agglutinate, it is the indicative of such blood group.

Antigen: a substance (a toxin or an enzyme) that stimulates an immune response in the body such
as production of antibodies.

Antibody: Any large variety of protein normally present in the body or produced in response to
an antigen which it neutralizes, thus producing an immune response.

APPARATUS AND REAGENT: White tile, blood lancet, alcohol swab, toothpick, monoclonal
antibodies A, B and D (O).

PROCEDURE

₰ Put on protective wears.

₰ Label white tile A, B and O according.
₰ Wipe the area of the fingertip to be pricked with an alcohol swab for the purpose of
sterilization.
₰ Squeeze the area and prick with lancet.
₰ Discard the lancet immediately in the sharp box.
₰ About 10uL blood collected is added to the A, B and O labeled tile.

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 ₰ A single drop of each of the Anti-A, B & D Monoclonal antibodies was then pipette and
applied to the first, second and third portion of the blood respectively.
₰ The monoclonal antibodies and the blood samples were carefully mixed.
₰ The tile was gently rocked observing any possible agglutination. The red blood cells
carrying one of these antigens are exposed to the corresponding antibodies.
₰ Formation of agglutination between blood sample and antisera confirms blood group.

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RESULT INTERPRETATION

Figure 17: Blood grouping

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2.3 NUTRITION
2.3.1 PROXIMATE ANALYSIS
Proximate analysis is a method for the quantitative analysis of the different macronutrients in
food based on the Weende analysis developed in 1860.Proximate composition is the term usually
used in the field of feed/food and means the 6 components of moisture, crude protein, ether
extract, crude fiber, crude ash and nitrogen free extracts, which are expressed as the content (%)
in the feed, respectively.

Figure 18: Proximate flow

Figure 19: Proximate composition

2.3.1.1 ASH CONTENT

Ash is the inorganic residue remaining after the water and organic matter have been removed by
heating in the presence of oxidizing agents, which provides a measure of the total amount of
minerals within a food.

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Ash is an inorganic residue remaining after the material has been completely burnt at a
temperature of 600℃ in a muffle furnace. It is an aggregate of non-volatile inorganic elements.

Analytical techniques for providing information about the total mineral content is based on the
fact that the minerals (the “analyte”) can be distinguished from all the other components (the
“matrix”) within a food in some measurable way. The most widely used methods are based on
the fact that minerals are not destroyed by heating, and that they have a low volatility compared
to other food components.

Ash contents of fresh foods rarely exceed 5%, although some processed foods can have ash
contents as high as 12%, e.g., dried beef.

DRY ASHING

Dry ashing is incineration at high temperature (525°Cor higher) accomplished in a muffle

furnace. The advantages of conventional dry ashing are that it is a safe method, it requires
nodded reagents or blank subtraction, and little attention is needed once ignition begins. A large
number of crucibles can be handled at once, and the resultant ash can be used for analyses like
individual elements, acid-insoluble ash, and water-soluble and insoluble ash. The disadvantages
are the length of time required (12 - 18 h, or overnight) and expensive equipment. There will be a
loss of the volatile elements and interactions between mineral components and crucibles.
Volatile elements at risk of being lost include arsenic, boron, cadmium, chromium, copper, iron,
lead, mercury, nickel, phosphorus, vanadium and zinc.

APPARATUS

Crucible (or similar porcelain or metal dishes), Muffled furnace, Sample, Hot plate, muffle
furnace.

PROCEDURE

1. Dry a representative sample (weigh accurately to nearest mg ~ 3–5 g of samples) in a crucible

in an oven at 130°C overnight. Char the sample on an electric hot plate or over a low flame in a
fume cupboard until it has ceased smoking.

2. Place the above crucibles (containing charred sample) in cold muffle oven and bring the
temperature to 550°C.

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3. Ignite the sample 12- 18 h (or overnight) at550°C.

4. Turn off the muffle furnace and wait to open until the temperature has dropped to at least
250°C, preferably lower. Open door carefully to avoid losing ash that may be light and fluffy.

5. Use safety tongs to quickly transfer the crucibles to a desiccator with a porcelain plate and
desiccant. Cover crucibles, close the desiccator, and allow crucibles to cool prior to weighing.

Calculation:

Weight of Ash = (Initial weight of crucible + sample)- (final weight of crucible + sample)

weight of ash
%ASH= × 100
weight of sample

2.4.1.2 CRUDE FIBRE

The dry, fat-free material is boiled successively with dilute acid and alkali for a specified time
period and filtered. The residue is dried and ignited. The loss in weight on ashing gives the crude
fiber. This consists chiefly of cellulose and lignocellulose.

APPARATUS & REGEANTS

Soxhlet apparatus, Conical flask, Gouch crucible, Dil. H2SO4 (0.255M), Dil. NaOH (0.313M),
Ethyl alcohol

PROCEDURE

• Defat the sample using soxhlet apparatus.

• Weigh 5g of defatted sample.
• Boil with 1.25% H2SO4, filter and Boil with 1.25% Na2SO4, filter
Add ethyl alcohol to reduce or completely remove the acidity and basicity.
• Dry residue.
• Place in the muffle furnace at 600°c for about 3hours.
• Allow to cool and weigh.

CALCULATIONS

Wt. of crude fiber = (initial Wt. of sample + crucible) - (final Wt. of sample + crucible)

%Fiber content = weight of fiber/weight of sample × 100

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2.3.1.3 MOISTURE CONTENT
DETERMINATION OF MOISTURE CONTENT BY OVEN DRYING METHOD (AOAC,
2005).

This method relies on measuring the mass of water and volatile substances in a known mass of
sample. The moisture content is determined by measuring the mass of a feed sample before and
after the water/volatile substances are removed by heating. The sample is heated under specified
conditions, and the loss of weight is used to calculate the moisture content of the sample. The
basic principle of this technique is that water/volatile substances have a lower boiling point than
the other major components within foods, e.g., lipids, proteins, carbohydrates and minerals.

Although the procedure is simple one theoretically, this is often extremely difficult to achieve in
practice because the high temperatures or long times required to remove all of the water
molecules could lead to changes in the mass of the food matrix, e.g., due to chemical changes of
some components

CALCULATION

Weight of moisture = (initial weight of Petri dish +sample) - (final weight of Petri dish + sample)

weight of moisture
%Moisture content= ×100
weight of sample

2.3.1.4 CRUDE FAT

The sample of dried foodstuff is placed in a continued extractor (Soxhlet) and subjected to
extraction with ether. The ether soluble substances thus removed are collected in a flask, dried ad
weighed. The materials extracted would include, besides the tri-glyceride, materials such as
phospholipids, sterols essential oils, pigments, waxes, etc., hence the term “crude fat”. If the
sample contains water-soluble sugars as in molasses, the weighed sample should be washed with
water and dried before extraction.

APPARATUS

Soxhlet Extractor: The extractor consists of the extraction flask, thimble, and condenser

PROCEDURE

In this method the sample is dried, ground into small particles and placed in a porous cellulose
thimble. The thimble is placed in an extraction chamber, which is suspended above a flask
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containing the solvent and below a condenser. The flask is heated and the solvent evaporates and
moves up into the condenser where it is converted into a liquid that trickles into the extraction
chamber containing the sample. The extraction chamber is designed so that when the solvent
surrounding the sample exceeds a certain level it overflows and trickles back down into the
boiling flask. At the end of the extraction process, which lasts a few hours, the flask containing
the solvent and lipid is removed. In some device a funnel allows to recover the solvent at the end
of the extraction after closing a stopcock between the funnel and the extraction chamber. The
solvent in the flask is then evaporated and the mass of the remaining lipid is measured. The
percentage of lipid in the initial sample can then be calculated

CALCULATIONS

Weight of fat=(weight of flask + oil)−(weight of flask )

weight of oil
%FAT = ×100
weight of sample

2.3.1.5 TOTAL PROTEIN CONTENT

Protein is the most important constituent of food and foodstuff.

Protein are compounds made up of amino acids.

‫ךּ‬ Weigh 0.5g of the sample and homogenize with 4.5ml of 0.4M phosphate buffer solution
in a crucible.
‫ךּ‬ Transfer the homogenate into a 5ml test tube.
‫ךּ‬ Centrifuge at 3200rpm for 5mins.
‫ךּ‬ Prepare 3tubes for the sample, in order of test, blank and standard.
‫ךּ‬ Pipette 20ul of supernatant into test and standard tubes.
‫ךּ‬ Add 1.0ul of biuret reagent in all.
‫ךּ‬ Add 20ul of sample into test, 20ul of standard reagent into standard and 20ul of water
into blank.
‫ךּ‬ Incubate on bench for 30minutes.
‫ךּ‬ Measure the absorbance at 546nm.
CALCULATION

O . D of sample
Absorbance= ×58.48
¿ O . D of standard

Absorbance
% of protein= ×100
Weight of sample

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2.3.1.6 CARBOHYDRATE CONTENT
This constituent is chiefly made up of starch and sugars. However, the direct determination is
difficult and time consuming. Hence, it is customary to derive this value by deducting the total of
crude protein, crude fat, crude fiber and total ash from 100

CALCULATION

Carbohydrate ( Nitrogen Free Extract ) =100-\{%moisture+%fat+%crude fiber+%protein\}

2.4 ISOLATION OF PHYTOCHEMICALS

2.4.1 QUANTIFICATION OF PHYTOCHEMICALS USING SOXHLET EXTRACTOR
A Soxhlet extractor is a piece of laboratory apparatus that was originally designed for the
extraction of a lipid from a solid material. Typically, a Soxhlet extraction is used when the
desired compound has a limited solubility in a solvent, and the impurity is insoluble in that
solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small
amount of solvent to dissolve a larger amount of material.

A soxhlet extractor consists of: the thimble, the siphon, the reflux condenser, the soxhlet
extractor, and the heating apparatus.

Figure 20: Soxhlet apparatus

To extract a substance from a compound, the materials needed are usually assembled in this
format:

1. The source material containing the compound to be extracted is placed inside the thimble.
2. The thimble is loaded into the main chamber of the Soxhlet extractor.
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 3. The extraction solvent to be used is placed in a distillation flask.
4. The flask is placed on the heating element.
5. The Soxhlet extractor is placed atop the flask.
6. A reflux condenser is placed atop the extractor.

2.4.1.1 PRINCIPLE OF OPERATION

The solvent is heated to reflux. The solvent vapour travels up a distillation arm and floods into
the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools,
and drips back down into the chamber housing the solid material. The chamber containing the
solid material slowly fills with warm solvent. Some of the desired compound dissolves in the
warm solvent. When the Soxhlet chamber is almost full, the chamber is emptied by the siphon.
The solvent is returned to the distillation flask. The thimble ensures that the rapid motion of the
solvent does not transport any solid material to the still pot. This cycle may be allowed to repeat
many times, over hours or days.

During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many
cycles the desired compound is concentrated in the distillation flask. The advantage of this
system is that instead of many portions of warm solvent being passed through the sample, just
one batch of solvent is recycled.

After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the
extracted compound. The non-soluble portion of the extracted solid remains in the thimble, and
is usually discarded.

2.4.1.2 APPLICATIONS OF SOXHLET EXTRACTION

1. EXTRACTION OF THE PHYTOCHEMICALS FROM PLANTS.

A rig is built using stands and clamps to support the extraction apparatus. Following this, the
solvent (250 ml of ethanol) is added to a round bottom flask, which is attached to a Soxhlet
extractor and condenser on a heating mantle. The crushed plant material is loaded into the
thimble, which is placed inside the Soxhlet extractor. The side arm is lagged with glass wool.
The solvent is heated using the heating mantle and will begin to evaporate, moving through the
apparatus to the condenser. The condensate then drips into the reservoir containing the thimble.
Once the level of solvent reaches the siphon it pours back into the flask and the cycle begins
again. The process is then allowed to run for several hours depending on the plant. Once the
apparatus has been set up the extraction it can be left to run without direct supervision. It is not
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advised to leave the equipment completely alone due to the mix of running water and an
electrical appliance, so a technician or other lab user should be made aware. The equipment can
be turned on and off when overnight running is not permitted, and the time split over a number
of days.

2. DETERMINATION OF FAT CONTENT

In this method the sample is dried, ground into small particles and placed in a porous cellulose
thimble. The thimble is placed in an extraction chamber, which is suspended above a flask
containing the solvent and below a condenser. The flask is heated and the solvent evaporates and
moves up into the condenser where it is converted into a liquid that trickles into the extraction
chamber containing the sample. The extraction chamber is designed so that when the solvent
surrounding the sample exceeds a certain level it overflows and trickles back down into the
boiling flask. At the end of the extraction process, which lasts a few hours, the flask containing
the solvent and lipid is removed. In some device a funnel allows to recover the solvent at the end
of the extraction after closing a stopcock between the funnel and the extraction chamber. The
solvent in the flask is then evaporated and the mass of the remaining lipid is measured. The
percentage of lipid in the initial sample can then be calculated.

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 Figure 22: Oven

Figure 21: Hot air oven

Figure 23: Organ harvest fromFigure

specimen
24: Freezer
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 Figure 35: Electrophoretic power pack

Figure 36: Electrophoretic chamber

Figure 37: Measuring cylinders Figure 38: Glassware

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Figure 43: pH meter

Figure 44: Water bath

Figure 45: Hot plate Figure 46: Spectrophotometer

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CHAPTER 3
3.1 CHALLENGES ENCOUNTERED DURING THE SIWES PROGRAM
The Nigerian institute of medical research (NIMR) had so much to offer, as it is an ideal
environment for research studies, although these challenges made our work harder:

1. Irregularity of power supply: although, the institution had a backup power supply, it
usually takes about a minute to become functional, during moments of power failure from
the state, which made power sensitive works such as centrifugation, weighing and so on
harder.
2. Dependency on samples from field work, performed by the institute as the populace in
Lagos were mostly unwilling to come to the institute.
3. Malfunctioning equipment posed a great problem, as it could sometimes lead to
frustration when carrying out experiments.
4. Inadequate, aged and unsophisticated equipment made research works tedious and as
such experimental results could not be relied upon

3.2 EXPERIENCE GAINED.

1. Management of the animal colony and care of the laboratory animal models.
2. Acquired the skills of blood and sample collection from laboratory rodents.
3. Learnt the process of drug administration and the methods of carrying out curative and
suppressive tests.
4. Proficiency in the preparation of thin and thick film from laboratory rodents for malaria
diagnosis
5. Ability to apply theoretical knowledge to practice.
6. Operation several specialized biochemical equipment, such as centrifuge,
spectrophotometer, Soxhlet apparatus, UV illuminator etc.
7. Adherence to strict protocols and ability to also, work effectively with minimal
supervision.
8. Ability to adjust to various working conditions in the laboratory.
9. Ability to identify sexes of laboratory animals

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 10. Working with NIMR staffs and other IT students widened my understanding, greatly
increased my interest in studying and gave me the ideal knowledge of team work. It has
also provided me the opportunity to meet and work with other scientists and researchers.

3.3 RECOMMENDATIONS.
3.3.1 RECOMMENDATIONS FOR THE COMPANY

NIMR is a great learning institution for interns. NIMR is a large and old medical institution and
helps interns improve and develops their skills. I would recommend NIMR to keep hiring interns
with different educational background, to help build and improve the institution and the country
as a whole with the knowledge they gained from their studies.

3.3.2 RECOMMENDATIONS FOR THE UNIVERSITY

The part that I found most interesting during my internship was the practical application of
theoretical works done school. During the period of my SIWES, I learned how to apply my
theoretical knowledge into real life situations and this provided me with an even better
understanding of what was learned in the four walls of the classroom.

Working for NIMR has changed my perceptions of the importance of biochemistry as a branch
of science & medicine, as it forms the bedrock through which all other biological sciences and
researches are formed.

That is why I would recommend my university to increase the teaching of practical applications
of topics learned in classes, as students learn better when they carry out practical on these
applications themselves. And also, the period of SIWES for the department of biochemistry
should elongated further by a month.

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CHAPTER 4
4.1 CONCLUSION
The three months plus a few weeks of my SIWES programme have been very instructive for me.
The Nigerian institute of medical research(NIMR) has offered me opportunities to learn and
develop myself in many areas. I gained a lot of experience, especially in the malaria research
sub-unit. A lot of the tasks and activities that I have worked on during my internship are familiar
with what I’m studying at the moment. I worked in many areas where I did different work. This
gave me the chance to find out which areas I want work in after my education. The area that I
found most interesting is malaria research &molecular biology.

As a bonus, I got to experience for the very first time the International scientific conference,
where important scientific discoveries made by scientists in the country were presented, medical
issues plaguing the country were addressed and possible solution were proposed.

There is a big difference between school theoretical works and the tasks and activities carried out
during the internship program. This internship was definitely an introduction to the actual work
field for me. I have learned to work in a scientific organization and applied my knowledge into
practice.

I learned a lot from the different interns that I worked with during my programme period. Each
intern had a different educational background and that made it interesting for me. By working
with them I got to learn from them, shared my knowledge with them and became aware of their
educational backgrounds.

My coordinators during my internship were Dr. A. O. Aina and Mr. Olusola Ajibaye, who were
fountains of knowledge during my internship. As professionals in pharmacology and
biochemistry respectively, they expanded my views and limited knowledge. They were very
helpful and always willing to dish out advice and feedback which I appreciated.

Interning at NIMR was definitely a learning experience. I had fun attending the scientific
conference and the presentations performed. I learned how to work alone, as well as with others.
This internship was definitely beneficial for me and I’m grateful and thankful that I got to
experience and learn many things.

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