DEPARTMENT OF BIOCHEMISTRY

FACULTY OF SCIENCES
BAYERO UNIVERSITY, KANO

STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

REPORT

BY

MAHMUD ABDULLAHI GINYA

SCI/08/BCH/000313

A PRE-REQUISITE FOR THE AWARD OF A GRADE IN SIWES SUBMITTED IN

PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE IN BIOCHEMISTRY

SUPERVISED BY
MALL. A.A MUHKTAR

CERTIFICATION
I hereby certify that the Students Industrial Work Experience Scheme (SIWES) report
submitted to the Department of Biochemistry, Bayero University Kano, as a requirement for
the award of degree of Bachelor of Science in Biochemistry, has been composed by myself
and has not been presented or accepted in any previous application for a degree. The work, of
which this is a record, has been carried out by myself unless otherwise stated and where the
work is mine, it reflects personal views and values. All sources of information have been
acknowledged by means of references including those of the Internet.

Mahmud Abdullahi Ginya

Mall. A.A Muhktar

Mall. Nasir Abdullahi

SIWES Coordinator

.
Signature/Date

..
Signature/Date

..
Signature/Date

DEDICATIONACKNOWLEDGEMENTS

TABLE OF CONTENTS
TITLE PAGEi
CERTIFICATION....................................................................................................................ii
DEDICATION.........................................................................................................................iii
ACKNOWLEDGEMENT........................................................................................................iv
LIST OF FIGURES...................................................................................................................v
GLOSSARY.............................................................................................................................vi
PREAMBLE
The Industrial Training Fund (ITF)...........................................................................................
Functions of the ITF

...........................................................................................................

The Students Industrial Work Experience Scheme (SIWES)...................................................

Objective of SIWES..................................................................................................................
Importance of SIWES................................................................................................................

CHAPTER ONE
Place of Industrial Training........................................................................................................
Brief History of the Organization..............................................................................................

CHAPTER TWO
EQUIPMENT, REAGENTS AND PREPARATION.........................................................
1.1

Equipments........................................................................................................
1.1.1

Ethylene Diamine Tetracetic Acid (EDTA) Bottles.........................................

1.1.2

Genotype Electrophoresis Machine...................................................................

1.1.3

Reflotron Machine.............................................................................................

1.1.4 Accu-check
1.1.5 Centrifuge Machine
1.1.6 Autoclave...
1.1.7 Incubator
1.1.8 Thermostatic Drying Oven
1.1.9 FACS Count Machine (CD4 Machine).

1.2

Reagents and their Preparation..

CHAPTER THREE
METHODS/ANALYSIS OF ROUTINE WORK DONE IN THE VARIOUS SECTION
OF THE LABORATORY
2.1

Clinical Chemistry Section...

2.2

Haematology Section...

2.3

Medical Microbiology Section.

2.4

Immunology Section

PREAMBLE

THE INDUSTRIAL TRAINING FUND (ITF)

The Industrial Training Fund was established by Decree No. 47 of 8th October, 1971, with
the aim of promoting and encouraging the acquisition of skills in industry and commerce
with a view to generating a pool of indigenous manpower sufficient to meet the needs of the
economy(ITF, 2010). This was the first of the three Manpower Training and Development
Agencies created by the Fedhjjeral Military Government during the Second National
Development Plan period (1970 1974).
After the establishment of agency, ITF rose to its challenges by organizing programs that
could among other things, expose students of Nigerian Universities and Polytechnics to
practical experience in their course of study and provide a means through which students
could meet as well as interact with industrial personnel.

Based on the foregoing, the student work experience programme which basically prepares
students for the world of work has become an innovative phenomenon in the process of
manpower development and training in Nigeria, hence, the introduction of the Students
Industrial Work Experience Scheme (SIWES) into the educational training system

FUNCTIONS OF ITF
Key functions of ITF include:

Encouraging greater involvement of employers, particularly small employers, in the

organization and development of training programmes and facilities including the
establishment of Group Training Schemes and Centres in certain areas of economic
activity;

Building of training facilities of its own, in identified areas of national need;

Organizing research and studies into training as a support to other activities of the Fund;

Establishing uniform Nation

country;

al Vocational Apprenticeship Training Scheme in the

Seeking to harmonize ITFs non-formal training programmes with the curricula of formal
educational institutions;

Bearing a proportion of the direct cost of on-the-job and off-the-job training of Nigerian
employees.

(ITF, 2010)

STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

The Students Industrial Work Experience Scheme (SIWES) is a Skill Training Programme
designed to prepare and expose students of Nigerian University to the industrial work
situation they are likely to encounter after graduation. The need for the 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. Therefore, the employers were of the opinion that the theoretical education in
higher institutions was not responsive to the needs of employers of labour.

As mentioned above, 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. It is an effort to bridge the gap existing between theory and practice of
engineering and technology, science, Agriculture, Medical, Management and other
professional educational programs in Nigerian institutions. The scheme is funded by the
Federal Government of Nigeria and jointly coordinated by the National Universities
Commission (NUC) and Industrial Training Fund (ITF).

OBJECTIVE OF SIWES

To provide students with an opportunity to apply their knowledge in real work situation,

thereby bridging the gap between theory and practice.

To expose students to work methods and techniques in handling equipment and

machinery that may not be available in their institutions.

To make transition from university to the world of work easier and thus enhance students
contact for later job placement after graduation.

To enlist and strengthen employers involvement in the entire educational process of

preparing university graduates for employment in industries.

To prepare students for the work situation they are to meet after graduation (ITF, 2010)

IMPORTANCE OF SIWES
SIWES have a lot of importance among which are:

It exposes students to real life situation, thus supplementing the theoretical lesson.
It helps to improve the quality of skilled manpower of the students.
It gives students practical knowledge of course of study.
It provides a forum for industries to evaluate prospective employers and gives feedback

to institutions.
It establishes a close collaboration between institutions and industries, a factor which is
essential for preparing student for the workforce.

CHAPTER ONE
THE HISTORICAL BACKGROUND OF UMARU SANDA NDAYAKO
HOSPITAL BIDA
Umaru Sanda Ndayako General Hospital Bida was established and commission by
His Excellency former head of the state and commander in chief of arm forces.
General Yakubu Gawon G.C.F.R. on the 28th day of March 2009.
The General Hospital also known as Umaru Sanda Ndayako is located along Minna
Road opposite Zamfara Mosques Bida, at the point of commission the hospital is well equipt
with hospital material and well qualified staff.

CHAPTER TWO
EQUIPMENTS, REAGENTS AND THEIR PREPARATION
2.0

EQUIPMENTS

The Equipment used in the laboratory throughout the SIWES period is as follows:
2.1.1

Ethylene Diamine Tetracetic Acid (EDTA) Bottles

Ethylene Diamine Tetracetic Acid (EDTA) bottles with purple caps, Serum Separation Tube
(SST) with (Red Cap,) lithium heparin Tubes (Green Cap), Erythrocyte Sedimentation Rate
(ESR) Tube (Black cap) and Sterile Universal Container (red and White Caps) as shown in
the figure 1.1 below

Fig 2.1: Ethylene Diamine Tetracetic Acid Bottles

2.1.2

Genotype Electrophoresis Machine

Fig 2.2: Genotype electrophoresis Machine

2.1.3

Reflotron Machine

Fig 2.3: Reflotron Machine

2.1.4

Accu-check

Fig
2.4: Accu
2.1.5

Centrifuge Machine

Fig 2.5: Centrifuge Machine

2.1.6

Autoclave

check

Fig 2.6:
Autoclave
2.1.7

Incubator

Fig 2.7: Incubator

2.1.8

Thermostatic Drying Oven

Fig 2.8: Thermostatic Drying Oven

2.1.9

FACS Count Machine (CD4 Machine)

Fig 2.9: FACS Count Machine (CD4 Machine)

Fig 2.10: Microscope

2.2

REAGENTS AND THEIR PREPARATION

Leishman's stain
Leishman's stain is used in microscopy for staining blood smears. It provides excellent stain
quality and is generally used to differentiate and identify leucocytes, malaria parasites, and
trypanosomas. It is based on a mixture of methylene blue and eosin.
Preparation
To make about 400ml:
Leishman stain powder..0.6g
Methanol (methyl alchohol....400ml
Note: The methanol MUST be water free
1.

Weigh the Leishman powder and transfer it to a completely dry brown bottle. Add a
few dry glass breads (to assist in dissolving the dye)

2.

Using a dry cylinder, measure the methanol and add this to the stain. Mix well at
intervals until the dye is completely dissolved. Warming the solution in a 37 0C water
bath will help the dye to dissolve.
Caution: Methanol is toxic and highly flammable; therefore handle it with care well
away from an open flame.

3.

Label the bottle and mark it Flammable and Toxic. Store it at room temperature in the
dark. When kept tightly stoppered, the stain is stable for several weeks. Moisture must
not be allowed to enter the stain.

For use: Filter 50-100ml of the stain into a stain dispensing container which can be closed
when not in use.
Sodioum citrate anticoagulant, 32g/l
To make 100ml
Tri-Sodium citrate, dehydrate3.2g
Distilled waterto 100ml
1.

Weigh the chemical and transfer it to a container pre-marked to hold 100ml (or to a
100ml volumetric flask).

2.

Add about half the water and mix until the chemical is completely dissolved.

3.

Add water to the 100ml mark and mix well.

4.

Label the container and store it at 2-80C. Discard the reagent if it becomes
contaminated (turbid appearance).

Reagent for Genotype Test

TRIS Boric Acid Buffer Salts: TBE or Tris/Borate/EDTA, is a buffer solution containing a
mixture of Tris base, boric acid and EDTA. In molecular biology, TBE and TAE buffers are
often used in procedures involving nucleic acids, the most common being electrophoresis.
Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. EDTA is a mixture of divalent cations, particularly of
magnesium (Mg2+). Because these ions are necessary co-factors for many enzymes, including
contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic
degradation (Wikipedia.org)
Preparation

The already prepared salt was dissolved in 500ml of distilled water

Normal saline
Preparation

Nacl salt was dissolved in 500ml of distilled water

Reagent for Blood Group Test

Anti sera
The reagents listed above have been already prepared by the manufacturer. They are stable
and valid when used before the expiry date and stored under their stated temperatures.
NUTRIENT AGAR
Nutrient agar is a microbiological growth medium commonly used for the routine cultivation
of non-fastidious bacteria. It is useful because it remains solid even at relatively high
temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly
visible as small colonies. . It is used for the sensitivity of bacterial colony. The medium is
best prepared from ready to use dehydrated powder, available from most suppliers of culture
mediea. The various component of nutrient agar solution is shown in the table below.
Table 1.1: Nutrient Agar Compositions
INGREDIENTS

gm/ltr

Agar

15.00

Peptone

5.00

Sodium chloride

5.00

Beef extract

1.50

Yeast extract

1.50

pH

7.4

Distilled water

1ltr

Preparation
28.0gm was dissolved in 1000ml distilled water. It was heat gentle to dissolve the medium
completely. Sterilized by autoclaving at 15 psi (1210C) for 15 minutes and was cool to 47C,
and mixed well before pouring plates.
Cystine Lactose Electrolyte Deficient Medium (CLED)
CLED Agar is a valuable non-inhibitory growth medium used in the isolation and
differentiation of urinary organisms. Because CLED is electrolyte deficient, it prevents the
swarming of Proteus species. Cystine promotes the formation of cystine-dependent dwarf

colonies. Lactose fermenters produce yellow colonies on CLED agar; non-lactose fermenters
appear blue. It has a pH of approximately 7.3 (Wikipedia.org). CLED agar contains:
Table 1.3: CLED Agar Composition
Content

Concentration

Bromo thymol blue

20mg/l

Beef extract

3g/l

L-cystine

128mg/l

Peptone
Agar

4g/l
15g/l

Lactose

10g/l

Hydrolysed Cassin

4g/l

Tryptone

4.0g/l

pH

7.3

Preparation
36 grams of powder was weighed and added to 1 litre of cold de-ionized water and
autoclaved at 121C for 15 minutes. Cool to 470C and was mixed thoroughly before pouring
plates. Stored at 2-80C, preferably sealed in plastic bags to prevent loss of moisture.
Salmonella Shigella (S.S Agar)
Table 1.4: Salmonella Shigella Agar Composition
Content
Beef extract

5.00

Animal tissue

5.00

Sodium citrate

10.00

Sodium thiosulphate

8.50

Agar

15.00

Lactose

10.00

Ferric citrate

1.00

Brilliant green

Preparation

Concentration gm/ltr

0.00033

Neutral red

0.025

pH

7.00

Bile salts mixture

8.50

63.0 grams of powder was weighed and added to one (1) litre of distilled water and allowed
to soak for 10 minutes, it was swirled to mix and then heat to the boiling with agitation to
dissolve the medium completely. It was allowed to cool to 47C and then mixed thoroughly
and poured into sterile petri plates. No autoclaving was needed for autoclaving may destroy
the selective of the medium.
MacConckey agar
The medium is best prepared from ready to use dehydrated powder, available from most
suppliers of culture media. The medium is usually used at a concentration of 5.2g in every
100ml distilled water. The various component of nutrient agar solution is shown in the table
below:
Table 1.5: MacConckey Agar Composition
Content

Concentration (g/l)

Peptone

20

Lactose

10

Bile salts

Sodium chloride
Neutral red

5
0.075

Agar

12

Ph

7.4

Preparation
52g of powder was suspended in 1 litre of distilled water. It was bring boil to dissolve
completely. Sterilized by autoclaving at 121 0C for 15 minutes. Poured into sterile petri plates
and then store in a cool dark place.
Table 1.5: Reagents for Glucose
Content

Concentration

Peroxidise (horse raddish)

3.2 U

3,3 ,5,5-tetramethylbenzidine (indicator)

72.6g

Glucose oxidase

Preparation

3.2 U

The reagent was already automated. Stored at 2 to 30C, this can be used up to the expiry
date printed on the pack of the container.
Table 1.6: Reagents for Glutamate Oxaloacetate (GOT), (aspartate aminotransferase)
Content
Pyruvate peroxidise
Peroxidise
Alanine sulfinate
Thiamine pyrophosphate
ketoglutarate
imidazole dihidrochloride
Buffer

Concentration
1.5 U
18 U
792g
1.49g
16.2g
16.4g

Preparation
The reagent was already prepared by the manufacturer. Stored at 2 to 30C, this is valid and
stable up to the expiry date given by the manufacturer.
Table 1.7: Reagents for Glutamate Pyruvate Transaminase (GPT), (Alanine amino transferase)

Content
Pyruvate peroxidise
Peroxidise
Alanine
Thiamine pyrophosphate
ketoglutarate
imidazole dihidrochloride
Buffer

Concentration
1.5 U
18 U
230g
1.49g
16.2g
16.4g

Preparation
The content is valid when stored at 2 to 30C and is used before the expiry date.

Table 1.11: Reagents for UREA

Content
Urease (jack beans)

Concentration
4.7U

Tetrachlorophenoltetrabromosulfophthalein
(indicator)

20.4g

Buffer

Preparation
The reagents were already prepared by the manufacturer and normally stored at 2 to 30.
Under this condition, the tests can be used up to the expiry date printed on the pack or
container.
Table 1.12: Reagents for Creatinine
Content
Creatinine iminohydrolase

Composition
1.38U

N-methylhydantoinase

0.108U

Carbamoyl Sarcosine hydrolyse

0.439U

Sarcosine oxidase

0.475U

Peroxidase

2.29U

Ascorbate oxidase

0.285U

ATP

82.8g

Hydrochloride (indicator)

19.5g

Buffer

Preparation
The reagents were already prepared by manufacturer. The test can be used up to expiry date
printed on the pack or container when stored at 2 to 30C.

Table 1.13: Reagents for Billurubin

Content
Dyphylline

Concentration
0.84mg

2-methoxy-4niyrophenyldiazoniumtetrafluoroborate

10.4g

Preparation
The reagents were already prepared by manufacturer. The test can be used up to expiry date
printed on the pack or container when stored at 2 to 30C.

Table 1.14: Reagents for Alkaline Phosphatase

Content
O-cresolphthalein phosphate
N-methyl-D-glucamine

Concentration
394g
1005g

Magnesium

0.56g

Preparation
The reagents were already prepared by manufacturer. The test can be used up to expiry date
printed on the pack or container when stored at 2 to 30C.
Table 1.15: Reagents for Sodium ions
Content
Uranyl acetate
Magnesium acetate

Concentration
2.1Mm
20Mm

Sodium standard

150mEq/L

Preparation
The reagents were already prepared by manufacturer. Reagent set were all stored at room
temperature (15-300C). The reagents are stable until the expiration date indicated on the label.

Table 1.15: Reagents for Bicarbonate ions

When reconstituted according to the direction, CO2 reagent contains the concentrations on the
table below:
Content
PEP
Magnesium Sulfate

Concentration
1.8Mm
10mM

NADH

0.40mM

MDH (porcine)

1,250U/L

PEPC (microbial)

200U/L

Sodium Oxamate

2.5mM

Buffer (pH)

7.0

Non-reactive fillers and Stabilizers

0.1%

Preparation
Reconstitute CO2 reagent with the 6ml of CO2-free water. Mix by inversion 5-6 times. Do not
shake. Reconstituted reagents stored in the closed tightly stoppered bottle are stable for 1 day
at room temperature (18-260C) and 7 days refrigerated (2-80C).

CHAPTER THREE
METHODS/ANALYSIS OF ROUTINE WORK DONE IN THE VARIOUS
SECTION OF THE LABORATORY
USGH laboratory is divided into five (5) main units, these are:

3.0

Haematology unit
Medical microbiology/parasitology unit
Chemical pathology unit
Immunology unit
Transfusion unit
CHEMICAL PATHOLOGY UNIT

This is a unit of the laboratory that deals with the metabolism of the constituents of the blood.
Activities carried out in this unit include:

Cleaning work surfaces

Performing daily cleaning of the Reflotron machine

Carrying out urinalysis using the combi 9

Spinning and separating serum for use in the centrifuge machine

Carrying out blood sugar test using the accu-chek

Carrying out liver profile test using the Reflotron machine or working reagents

Carrying out electrolyte test using the Reflotron for potassium and creatinine or working
reagents for all electrolytes

3.1

Registering all tests carried out in their respective ledgers

COLLECTION OF BLOOD SAMPLES AT PHLEBOTOMY

Factors regarding the collection of blood specimens that can affect the correctness of clinical
chemistry test results include:
- Incorrect venepuncture technique
- Haemolysis of red cells,
- Collection into the wrong container
- Instability of some chemical substances in whole blood, serum, and plasma

VENEPUNCTURE TECHNIQUE
The following precautions need to be followed when collecting venous blood in chemical
pathology:
*

Do not apply the tourniquet too tightly or for too long a period because this will cause
venous stasis leading to a concentration of substance in the blood such as
haemoglobin, plasma proteins, potassium and calcium.

Do not collect the blood from an arm into which an intravenous (IV) infusion is being
given.

If an anticoagulated specimen is required, add the correct amount of blood with the
anticoagulant by gently inverting the container several times.

Follow a safe technique and wear protective gloves.

AVODING HAEMOLYSIS
The haemolysis (rupture) of red cells can be a serious source of unreliable test results. If red
cells are haemolysed, substances from the cells are release into the serum or plasma leading
to a false increase in the concentration of analytes, e.g. potassium. Haemolysis also interferes
with many chemical reactions.
Haemolysis can be avoided by:
*

Checking that the syringe and needle are dry and that the barrel and plunger of the
syringe fit well.

Not using needle with too fine a bore.

Not withdrawing the blood too rapidly or moving the needle once it is in the vein.
Frothing of the blood must be avoided.

Removing the needle from the syringe before dispensing the blood into the specimen
container. Allow the blood to run gently down the inside wall of the container.

Adding the correct amount of blood to anticoagulant. Do not shake the blood but
gentle mix it with the anticoagulant.

Using clean dry tubes bottles for the collection of blood from which serum is required
and by allowing sufficient time for the blood to clot and clot retraction to take place.
Red cells are very easily haemolysed by rough use of an applicator stick to dislodge a
clot.

Centrifuging blood samples for a minimum period of time. Centrifuging for 5 minutes
at about 1000g is adequate to obtain serum or plasma.

Not storing whole blood samples in, or next to, the freezing compartment of a
refrigerator.

SPECIMEN CONTAINERS AND ANTICOAGULANT

Specimen containers for clinical chemistry tests must be leak proof and chemically clean.
They should be well washed with detergent, rinsed in several changes of clean water before
being allowed to dry.
To avoid the confusion which often arises from the use of several different types of container,
the following system is recommended:
Non-urgent blood tests: Dispense about 5ml of blood into a dry tube or bottle and allow
clotting. When the clot has retracted, centrifuge the specimen and transfer the serum to a
labelled container. Tightly cap the container to avoid water evaporating from the specimen in
dry warm conditions or moisture entering in humid environments.
Urgent blood tests and paediatric blood tests: Dispense the correct amount of blood into a
tube containing lithium heparin anticoagulant. Gently mix the blood with the anticoagulant.
Centrifuge the specimen and transfer the plasma to a labelled container.
# Lithium heparin does not interfere with most chemical reactions and helps to minimize
haemolysis. It does not contain sodium or potassium. Anticoagulants such as EDTA and
fluride-oxalat contain sodium or potassium salts and therefore these cannot be used when
electrolytes are to be measured.
Blood glucose (non-urgent or urgent): Dispense the correct amount of blood into a tube
containing fluoride-oxalate. Gentle mix the blood with the anticoagulant. Centrifuge the
specimen to obtain plasma.
# Fluoride is an enzyme inhibitor. It prevents the breakdown of glucose to lactic by acid by
enzyme action (glycolysis). Blood collected into fluoride-oxalate can also be used for
measuring protein, urea and bilirubin but not for electrolytes or enzymes. Fluoride-oxalate
must not be used if measuring glucose using a reagent strip test.
STABILITY OF ANALYTES IN BLOOD SPECIMENS
Biochemical substances in blood are found in both plasma and cells but not necessarily in
equal concentrations:
More concentrated in plasma

Potassium
Phosphate
Aspartate amino
Transferase (AST)

More concentration in plasma

Lactate dehydrogenase
Sodium
Chloride

More concentration in plasma

Carbon dioxide
Creatinine
Glucose
Urea

Urate
Some of the chemical changes which may occur in blood specimens within a few hours of
being collected include:
*

Diffusion of potassium and some enzymes through the red cell membrane into the
serum or plasma.

Diffusion of carbon dioxide off the surface of the blood, leading to a lowering in the
concentration of bicarbonate with a compensatory increase in plasma chloride.

Decrease in the concentration of glucose by glycolysis (when fluoride-oxalate is not

used).

Reduction or losses in the activity of certain enzymes, for example acid phosphatise.

Decomposition of bilirubin in daylight or fluorescent light.

Some of these changes can be prevented by separating the plasma or serum forms the red
cells as soon as possible (within 1 hour) after the blood has been collected. The blood should
not be refrigerated before the serum or plasma is separated because this can lead to falsely
raised potassium values. Glycolysis can be prevented by using fluoride-oxalate anticoagulant.
The decomposition of bilirubin can be avoided by protecting specimens from light.
To minimize any alteration in the concentration of substances due to the ingestion of food or
day time variation, most blood specimens are best collected at the beginning of the day before
a patient takes food.
3.1.1

ELECTROLYTES TEST

Electrolytes are substances that become ions in solution and acquire the capacity to conduct
electricity. The electrolytes that are measured include sodium, potassium, bicarbonate and
chloride using their various working reagents.
Sodium Test
Sodium is the major cation of extracellular fluid. It plays a central role in the maintenance of
the normal distribution of water and the osmotic pressure in the various fluid compartments.
The main source of body sodium is sodium chloride contained in ingested foods. Only about

one-third of the total bodys sodium is contained in the skeleton since most of it is contained
in the extracellular body fluids.
Principle
Sodium is precipitated as the triple salt sodium magnesium uranyl acetate; the excess
uranium then reacts with ferrocyanide to form a coloured complex whose absorbance varies
inversely as the concentration in the test specimen.
Procedure
Filtrate preparation

Test tubes were labelled blank, standard and sample.

1.0ml of filtrate reagent was pipette to all tubes.

0.05ml of sample was added to all tubes and distilled water to the blank.

Tubes were vigorously shake and mixed continuously for 3 minutes.

All the tubes were centrifuged at high speed (1,500G) for 10 minutes after which their
supernatant fluids were tested as describe below:

Colour development

The test tubes were labelled corresponding to the above filtrate tubes.

1.0ml of acid reagent was added to all tubes.

0.05ml of each supernatant was added to their respective tubes and mixed.

0.05ml of colour reagent was added to all tubes and mixed.

Distilled was used to zero the spectrophotometer at 550nm.

Absorbencies of all tubes were red and recorded.

Calculations
Abs of Blank Abs of Sample
Abs of Blank Abs Standard
Reference Range
135 155mEq/L

X Conc. of Std (mEq/L) = Conc. of Sodium

Potassium Test
Potassium is the principle cation of the intracellular fluid. It is also an important constituent
of the extracellular fluid due to its influence on muscle activity. Its intracellular function
parallels that of its extracellular function, namely influencing acid-base balance and osmotic
pressure, including water retention.
Elevated potassium levels (hyperkalemia) are often associated with renal failure, dehydration
shock or adrenal insufficiency. Decrease potassium levels (hypokalemia) are associated with
malnutrition, negative nitrogen balance, gastrointestinal fluid losses and hyperactivity of the
adrenal cortex.
Principle
The amount of potassium is determined by using sodium tetraphenylboron in a specifically
prepared mixture to produce a colloidal suspension. The turbidity of which is proportional to
potassium concentration.
Procedure

Test tubes were labelled blank, standard and sample.

1.0ml of potassium reagent was pipette to all tubes.

0.01ml of samples was added to sample, standard reagent to the standard and mixed then
was allowed to stand at room temperature for 3 minutes.

Reagent Blank was used to zero the spectrophotometer at 500nm.

Absorbencies of all tubes were red and recorded.

Calculations
Abs of Unknown
Abs of Standard

X Conc. of Std (mEq/L) = Potassium Conc. (mEq/L)

Reference Range
3.4 5.3mEq/L
Chloride Test
Chloride, a major anion, is important in the maintenance of the cation/anion balance between
intra and extra-cellular fluids. This electrolyte is therefore essential to the control of proper
hydration, osmotic pressure, and acid/base equilibrium. Low serum chloride values are fond

with extensive burns, excessive vomiting, intestinal obstruction, nephritis, metabolic acidosis,
and in addisonian crisis. Elevated serum chloride value may be seen in dehydration,
hyperventilation, congestive heart valve, and prostatic or other types of urinary obstruction.
Principle
Hg(SCN)2 + 2Cl-

HgCl2 + 2SCN-

3SCN- + Fe3+

Fe(SCN)3 (red complex)

Chloride ions form a soluble, non-ionized compound, with mercuric ions and will displace
thiocyanate ions from non-ionized mercuric thiocyanate. The released thiocyanate ions react
with ferric ions to form a colour complex that absorbs light at 480nm. The intensity of the
colour produced is directly proportional to the chloride concentration.
Procedure

Test tubes were labelled blank, standard and sample.

1.5ml of chloride reagent was pipette to each tube.

0.01ml of sample was added to sample tube, standard reagent to standard and distilled
water to blank and mixed.

Tubes were incubated at room temperature for at least 5 minutes

Spectrophotometer was set at 480nm and was zero with reagent blank.

Absorbencies of all tubes were red and recorded.

Calculations
Abs of Unknown
Abs of Standard

X Conc. of Std (mEq/L) = Chloride Conc. (mEq/L)

Reference Range
98 106mEq/L
3.1.2

UREA TEST

Urea is the major end product of protein nitrogen metabolism. It is synthesized in the liver
from ammonia, which is produced by amino acid determination. The determination of serum
urea renal function or increased tissue protein breakdown is associated with increased Urea
Nitrogen levels. Whereas liver damage or pregnancy are associated with decreased levels.

Principle
The Berthelot reaction has long been used for the measurement of urea and ammonia. The
present method is a modified Berthelot.
Urea + H2O

urease

2NH3 + CO2

NH3 + Salicylate + Hypochlorite

2,2-Dicarboyindophenol

The BUN colorimetric procedure is a modification of the Berthelot reaction. Urea is

converted to ammonium by the use of urease. Ammonium ion then reacts with a mixture of
salicylate, sodium nitroprusside, and hypochlorite to yield a blue-green chromophore. The
intensity of the colour formed is proportional to the urea concentration in the sample.
Procedure

BUN Enzyme Reagent was reconstituted according to the instruction

1.5ml of BUN Enzyme Reagent was pipette into labelled test tubes. It was allowed to

equilibrate to room temperature.

0.01ml (10) of each sample was added to its respective tubes and then mixed gentle.

Deionized water was used as the sample for the Reagent Blank.
The tubes were incubated for five minute at 37 oC or ten minutes at room temperature

(18-30oC).
1.5ml of BUN Colour Developer was added and then mixed gently.
Tubes were incubated for five minute at 37oC or ten minutes at room temperature.
Reagent Blank was used to zero the spectrophotometer at 630nm.
Absorbencies of all tubes were red and recorded.

Calculations
Abs of Unknown
Abs of Standard

X Conc. of Std = Urea Nitrogen (mg/dL)

Reference Range
7-23mg/dL
3.1.3

CREATININE TEST

Creatinine is a Nitrogenous waste product formed from the metabolism of creatinine in

skeletal muscle. Creatinine diffuses freely throughout the body water. It is filtered from the
extracellular fluid by the kidney and excreted in the urine. The excretion of creatinine is
mainly renal and its elevation is highly specific for kidney diseases.
Principle
alkali

Creatinine + Sodium Picrate

Creatinine + Picrate complex

Creatinine reacts with picric acid in alkaline conditions to form a colour complex, which
absorbs at 510nm. The rate of formation of colour is proportional to the creatinine
concentration in the sample. In the endpoint method the difference in absorbance
measurements after colour formation yields a creatinine value corrected for interfering
substances.
Procedure

Equal volumes of creatinine picric acid reagent and creatinine buffer reagent were
combined and mixed well.

Test tubes were labelled blank, standard and sample

3.0ml of working reagent was pipette into the tubes

0.1ml of sample was transferred to its respective tube, standard reagent to the standard
and distilled water to reagent blank and mixed.

Tubes were incubated at 37oC for 15 minutes.

Reagent Blank was used to zero the spectrophotometer at 510nm.

Absorbencies of all tubes were red and recorded.
Absorbencies of all tubes were red and recorded.

Calculations
The creatinine value of the unknown is determined by comparing its absorbance change with
that of a known standard.
Abs of Unknown
X Conc. of Std = Conc. of Creatinine
Abs of Standard
Reference Range
Male: 0.9-1.50mg/dl
Female: 0.7-1.37mg/kl

3.1.4

UREA AND CREATININE USING REFLOTRON MACHINE

Urea samples were collected for the evaluation of renal function. The test is often used
together with the determination of Creatinine for differentiation between pre-renal and postrenal azotaemia on the basis of the urea/Creatinine ratio. These are products of

osmoregulation and are analyzed using the Reflotron. The reference ranges for urea and
Creatinine is 10 50mg/dl and 0.5 1.1mg/dl respectively.
Test Principle
After application to test strip, the sample flows into the reaction zone, in the case of blood
after separation of the erythrocytes from the plasma. The urea contained in the sample is
hydrolyzed to ammonium carbonate from which ammonia is expelled due to the alkaline
buffering. This leads to the partial colour change of a buffered indicator to green /blue, the
intensity of which is proportional to the urea concentration of the sample:
(NH2)2 CO + H2O

Urease

NH3 + indicator (yellow)

2NH3 + CO2
NH4+ + indicator (blue)

At a temperature of 37OC, the depth of colour formed is measured at 642nm and the urea
concentration displayed after about 190 seconds in mg/dl depending on how the instrument
has been set.
Sample Collection
Blood sample was collected in the Lithium Heparin tubes or Ethylene Diamine tetra acetic
tube.
Testing Procedure
Additional materials required are Reflotron instrument; Reflotron pipette and pipette tips or
capillary pipette and controls. The procedure used is as follows:

The instrument (Reflotron) was switched on.

When the display shows ready, the test strip was removed from the container
The strip was unwrapped, taking care not to bend it.
Using the Reflotron pipette, sample material was drawn into the pipette (avoiding
bubbles) and applied a drop to the centre of the red application zone carefully in order
not touch the application zone with the pipette tip. (The required volume of specimen for

application was 30nl).

With the sliding cover or flap opened, the test strip was placed on the guide within 15 sec
and slide forward horizontally until it locks into place, and the sliding cover/flap was

then closed.
The instrument displays urea/Creatinine to confirm that it has correctly read in the testspecific magnetic code. The display shows the number of seconds left before the result

was displayed. The concentration was calculated automatically from the readings taken
using a function and conversion factors that were entered in the instrument via the
magnetic strip on the underside of each test strip. The concentration was displayed in
mg/dl or mmol/l depending on whether the instrument has been set to show conventional

or S.I units.
The test strip used was then removed from the Reflotron and disposed of according to
laboratorys procedure.

3.1.5

LIVER FUNCTION TEST (LFT)

The liver function test was carried out using the Reflotron. The machine determines the
concentration of particular enzyme and proteins in the liver by the use of strips. Liver
function test comprises of the Total Billurubin, glutamate oxaloacetate transaminase (GOT),
Glutamate Pyruvate transaminase (GPT), and Alkaline phosphate (ALP).
Glutamate Oxaloacetate Transaminase (GOT)
Glutamate oxaloacetate transaminase (aspartate aminotransferase) belongs to the group of
transaminase which catalyze the conversion of amino acids to the corresponding -keto acids
and vice versa by transfer of amino groups. Aspartate aminotransferase is found in many
tissues of the body.
Test Principle
After application to the test strip, the sample flows into the reaction zone, in the case of blood
after separation of the erythrocytes from the plasma. In the presence of GOT, -ketoglutarate
and alanine sulfinate are converted to Pyruvate oxidase, the resulting Pyruvate is cleaved into
acetyl phosphate, carbon dioxide and hydrogen peroxide. In the presence of POD the
hydrogen peroxide converts an indicator into its oxidised blue form:
-ketoglutarate + alanine sulfinate
Pyruvate + PO43- + O2 + H20 PyOD
H2O2 + indicator (red)

POD

GOT

glutamate + Pyruvate + SO32-

acetyl phosphate + H202 + CO2

indicator (ox.) + H2O

Endogenous Pyruvate was eliminated in a preliminary reaction.

At a temperature of 370C the formation of the dye was measured kinetically at 567nm as a
measure of the enzyme activity of GOT and the result was displayed after 124 seconds. The
result displayed for a temperature of 370C, 300C or 250C and in u/l.
Glutamate Pyruvate Transaminase
Glutamate pyruvate transaminase (alanine aminotransferase) belongs to the group of
transaminase which catalyzes the conversion of amino acids to the corresponding -keto
acids and vice versa by transfer of amino groups.
Test Principle
After application to the test strip, the sample flows into the reaction zone, in the case of blood
after separation of erythrocytes from the plasma. In the presence of GPT, -ketoglutarate and
alanine are converted to glutamate and pyruvate. In the second reaction step, catalyzed by
pyruvate oxidase, the resulting pyruvate was cleaved into acetyl phosphate, carbon dioxide
and hydrogen peroxide. In the presence of POD the hydrogen peroxide converts an indicator
into its oxidised blue form:
-ketoglutarate + alanine

GPT

Pyruvate + PO43- + O2 + H2O

H2O + indicator (red.)

POD

glutamate + pyruvate
PyOD

indicator (ox.) + H2O

indicator (ox.) + H2O

Endogenous pyruvate was eliminated in preliminary reaction.

At a temperature of 370C the formation of the dye is measured kinetically at 567 nm as a
measure of the enzyme activity of GPT and the result displayed after 140 seconds. The result
displayed for temperature of 370 C , 300C or 25oC and in u/l
Alkaline Phosphatase (ALP)
Alkaline Phosphate (ALP) is present in various human tissues e.g. liver, bone, intestine and
placenta.
Test Principle
After application to the test strip, the sample flows into the reaction zone, in the case of blood
after separation of the erythrocytes from the plasma. ALP hydrolyzes o-cresol-phthalein and
transfers the phosphate group to the acceptor molecule methylglucamine. The coloured

hydrolysis product o-cresolphthalein that is produced per unit of time under alkaline
conditions is directly proportional to alkaline phosphate activity:
O-cresolphthalein phosphate + methylglucamine

o-cresolphathlein + methylglucamine

phosphate.
Dye formation is determined kinetically at 37 0C as a measure of the enzyme activity of ALP.
The result is displayed after approximately 135 seconds in U/l.
Sample collection for LFT
Blood sample was collected into the lithium heparin tube or Ethylene diamine tetra acetic
tube.
Testing procedure for LFT (GOT, GPT, ALP)
Additional material required (not supplied): Reflotron instrument; Reflotron pipette and
pipette tips or capillary pipette, controls and usual laboratory equipment were used for
collecting blood. The procedure used is as follows:

The instrument was switched on

When the display showed READY, the test strip was removed from the container.
The strip was unwrapped carefully so as not to bend it.
Using the Reflotron pipette, the sample material drawn into the pipette (avoiding bubbles)
and applied as a drop to the centre of the red application zone, taking care not to touch the
application zone with the pipette tip. Required volume of specimen

for

application

was 30nl
With the sliding cover or flap open, the test strip was placed on the guide within 15

seconds and slide forward horizontally until it locks intro place

The sliding cover/flap was then closed.
The instrument displays ALP to confirm that it has correctly read in the test-n
magnetic code. The display shows the number of seconds left before the

specific

result

was

displayed.
ALP activity was calculated automatically from the readings taken using function and
conversion factors that are entered in the instrument via the magnetic strip on the

underside of each test strip. The enzyme activity is shown in U/l

The test strip used was then removed from the Reflotron and disposed of according to
your laboratorys procedure. The table below shows the reference range for male and
female:
Table 3.3: Reference Range for Male and Female

Component
Glutamate pyruvate transaminase(GPT)
Alkaline Phosphate(ALP)
Total Billurubin
Glutamine Oxaloacetate Transaminase (GOT)

3.2

Male (mg/dl)
Up to 41
Up to 270
0.5-1.0
Up to 40

Female (mg/dl)
Up to 32
Up to 240
0.5-1.0
Up to 33

HAEMATOLOGY UNIT

Haematology is unit in the main laboratory which is concerned with the study of blood, the
blood forming organs and blood diseases. The activities carried out in this unit include:

Cleaning the work bench with normal saline and methylated spirit

Making forms for the patients tests

Running full blood count using stained film on microscope and counting chamber

Making thin slides of blood films

Staining the slides with liechmans stain

Carrying out malaria parasite tests using MP strip

Viewing the stained films under microscope for detection of malarial parasites

Carrying out erythrocyte sedimentation rate ESR test

Carrying out genotype test using the cellulose acetate electrophoresis technique

Carrying out clotting profile test

Registering the results onto the ledger and the forms

2.4 MEDICAL MICROBIOLOGY UNIT

The medical microbiology section deals with the study of organisms of microscopic size,
including bacteria, protozoan, viruses, and certain algae and fungi. The unit is divided into
two; microbiology section and parasitology
The medical microbiology section has media room as well as general microbiology section
where basic tests are being carried out.
Media Room

The media room is where all the media used for culturing are prepared and stored. The most
common media used in the laboratory are; nutrient agar, chocolate agar, Cystine Lactose
Electrolyte Deficiency (CLED) agar, Salmonella Shigella Agar (SSA) and Mac Conkey agar
Activities carried out in this unit include:

Cleaning my work surfaces every beginning and ending of work

Drying culture and sensitivity plates in the incubator

Reading sensitivity plates

Culturing samples like stool, urine, sputum, and swab

Reading the cultured plates

Carrying out seminal fluid analysis SFA using the sperm quality analyser

Carrying out microscopy of various samples

Carrying out tests like the hepatitis b surface antigen and hepatitis c virus tests
respectively

Running tests like the widal, pregnancy

Performing tests like the VDRL

Preparation of media using the autoclave

Registering all results of tests carried out for future reference

2.5 IMMUNOLOGY UNIT

This is a unit of the laboratory where the immune system of HIV patients is assay and
counted using the BD FACSCount machine (CD4 Machine). The activities carried out in this
unit include:

Cleaning the work bench with jik and methylated spirit to disinfect it

Giving the samples numbers

Cleaning the FACSCount machine

Preparing the samples using the appropriate reagents

Running the samples on FACSCount machine

Cleaning the FACSCount machine after running the samples

Shutdown the machine

Registering all results of tests carried out for future reference

2.6 TRANSFUSION UNIT

This is a unit of the laboratory where blood samples are screen for infectious diseases before
been transfuse into the patient. The activities carried out in this unit include:

Cleaning work surfaces

Carrying blood grouping test and cross matching

HIV screening test

HIV Counselling

Registering all results of tests carried out for future reference

ANALYSIS
3.0

CHEMICAL PATHOLOGY UNIT

IMMUNOLOGY UNIT
This is a unit of the laboratory where the immune system of HIV patients is assay and
counted using the BD FACSCount machine (CD4 Machine).
SPECIMEN COLLECTION AND STORAGE
FACSCount technique is designed to be used with human whole blood samples. Whole blood
is collected in a K3 EDTA vacutainer tube and stored no longer than 6 hours at room
temperature (20-250C).
Test procedure
All reagents were brought to ambient temperature
Procedure for preparing and running controls
Normal blood is needed to run controls. Blood sample must be collected in a K3 EDTA
vacutainer tube from a normal donor and run immediately or could be stored no longer than 6
hours at room temperature (20-250C).
Controls are prepared by adding normal blood, then fixative solution to the CD4 and CD8
reagents tubes followed by addition of control beads before reagent tubes are run. Controls
beads are packed in tube pairs similar to reagent tube pairs.
The normal blood donor was placed in the workstation. Two reagent tube pairs from foil bag
were removed. The foil bag was sealed and the unused reagent pairs were returned to the
refrigerator.

The tabs of the two reagent tube pairs were labelled:

Pair one:

Label the CD4 (green top) Zero

Label the CD8 (clear top) Low

Pair two:

Label the CD4 (green top) Medium

Label the CD8 (clear top) High

The pairs were vortex upside down for 5 seconds. The pairs were vortex upright for 5
seconds.

The reagent tubes was opened with coring station and closed to protect the reagents
from light.

The whole blood was mixed by inverting the tube five times.

50l of normal whole blood was pipette into each of the four reagent tubes. Each was
changed between tubes.

Tubes were caped and vortex for seconds.

Tubes were incubated for 60 to 120 minutes at room temperature (20-250) in the dark.

After which tubes were uncapped and 50l of fixative solution was pipette into each
reagent tube. Tips were changed between tubes.

Reagent tubes were recapped with new caps and vortex upright for 5 seconds.

Incubated for at least 30 minutes at room temperature in the dark.

One pair of Zero/Low control beads and one pair of Medium/High control beads were
removed from the control kit and placed in the control area of the workstation.

Tubes labelled zero, low, medium and high were uncapped.

The zero/low control beads pair was vortex for 6 seconds and reverse pipette of 50 l
of low control beads into the tube labelled low.

The medium/high control bead pair was vortex for 6 seconds and reverse pipette of
50l of medium control beads into the tube labelled medium.

Reverse pipette of 50l of the high control beads into the tube labelled high.

The controls were then run on the BD FACSCount Machine within 24 hours of adding
the control beads.

Procedure for preparing and running controls

Patient blood sample must be collected in a K3 EDTA vacutainer tube from a normal donor
and run immediately or could be stored no longer than 6 hours at room temperature (20250C).

Patient samples were prepared by adding blood, then fixative solution to the CD4 reagent
tube.
The normal blood donor was placed in the workstation. One reagent tube pair was removed
for each patient. The foil bag was sealed and the unused reagent pairs were returned to the
refrigerator.

The tabs of one reagent tube pair was labelled with the patient laboratory number
The pairs were vortex upside down for 5 seconds. The pairs were vortex upright for 5
seconds.

The reagent tubes were opened with coring station. The workstation was closed to
protect the reagents from light.

The patient whole blood was mixed by inverting the tube five times

50l of whole blood was pipette into each two reagent tubes.

The tubes were capped and vortex for 5 seconds.

Then incubated for 60-120 minutes at room temperature (20-25 0) in the dark. The
reagent pairs were placed in the workstation and the cover was closed to protect the
reagents from the light.

The tubes were uncapped and 50l of fixative solution was pipette into each reagent
tube.

The reagent tubes were recapped with new caps and were vortex upright for 5
seconds.

The tubes were run on the FACSCount Machine within 48 hours of preparation.
Samples were stored at room temperature in the workstation until they were run on
the Machine. They were vortex upright for 5 seconds immediately before running.