STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

CONDUCTED AT

SPEACIALIST HOSPITAL SOKOTO

AND AT

GENERAL HOSPITAL ALIERO

BY

ABDULRAHMAN SANI ALIERO

ADM NO: 1310213001

SUBMITED TO THE DEPARTMENT OF BIOLOGICAL SCIENCE,

FACULTY OF SCIENCE, KEBBI STATE UNIVERSITY OF SCIENCE AND

TECHNOLOGY ALEIRO, KEBBI STATE.

APRIL, 2015
 DEDICATION

This report is dedicated to my beloved parents and siblings.

 ACKNOWLEDGEMENT

I remain grateful to Almighty Allah for His infinite mercies, love and kindness towards me and
for making this industrial training a reality.

Also, I want to appreciate the contribution of my beloved parents my elder sister and my
siblings for their immense support and relentless effort in making my educational career a
success.

My profound gratitude also goes to my Immediate supervisor in the Laboratory; at uduth

and at specialist HOD of uduth microbiology laboratory Mal. Mahdi Adamu, Sabiu, Victor,
Sabastain, Jonathan, Aisha, Franciscar Mal. Suleiman Iye, Mrs Onma Okpoju, Mrs Suwaiba
Ibrahim, Regina who had played the role of a supervisor, advocate, counselor. I am indebted to
you all, for your constructive ideas, useful suggestion, encouragement and your ever willingness
to assist me in this industrial training. May Allah reward you abundantly and crown all your
endeavors with success.

I wish to extend my special thanks to all my friends Adetola Akanji aka Mamoo, Aliyu
Mohd Aliyu, Dinah Luka Zanga, Eunice Abidakun, Pamela .M. Isha, Abdulhamid Zubairu aka
confirm, Jerry Billyaminu. For their relentless effort during my industrial training and the rest
that I did not mention their names. May God bless you all.

I also extend my profound gratitude to all well-wishers, may the Almighty God reward
you all (Amen)
TABLE OF CONTENTS

INTRODUCTION TO THE TRAINING

CHAPTER ONE

 Introduction to Microbiology Unit

 Reception section
 Sample collection and measure to be taken during processing
 Equipments used in microbiology laboratory
 Common laboratory reagents
 How to prepare media and sterilization

CHAPTER TWO - BACTERIOLOGY SECTION

 Gram staining
 Culture of Microorganism
 Sensitivity test

CHAPTER THREE – PARASITOLOGY SECTION

 Urine microscopy
 Stool microscopy
 Malaria parasite microscopy

CHAPTER FOUR - SEROLOGY

 Widal test
 Hepatitis test
 HIV test
 Conclusion
 CHAPTER ONE

1.0 INTRODUCTION

1.1 HISTORY OF SIWES

The Student’s Industrial Work Experience Scheme was established in 1973 by the

Industrial Training Fund (ITF). The SIWES is a programme designed to combat the problem of

inadequate skills necessary for employment of Nigerian graduates in industries. The scheme

therefore helps students acquire relevant practical skills alongside the theoretical ones that are

being taught in schools. The SIWES had headquartered in Jos, Plateau State and has area offices

across other states of the Federation.

The SIWES was integrated into the tertiary institutions syllabus by the Industrial Training

Fund (ITF) so as to check over-dependence on foreign expertise and reduce it to a level with

competent manpower that will make use of specialized skills to manage the major sectors of the

nation’s economy. Therefore, it provides students with the necessary experience that prepares

them for future challenges and thus makes them useful for themselves and the community.

Moreover, participation in the SIWES programme has become an imperative requirement

for the award of Diploma or Degree in various disciplines of most institutions of higher learning

within the country in tandem with the educational policy of the government. Participants of the

SIWES programme include undergraduate students of Pure and Applied sciences, Engineering

technology, Agriculture, Environmental Sciences, Education and Medical Sciences. The SIWES

programme is funded by the Federal Government of Nigeria, operated by ITF and coordinated by

National Universities Commission (NUC), National Commission for Colleges of Education

(NCCE), National Board for Technical Education (NTBE), Employers of Labor and various

institutions.

1.1.1 AIMS OF SIWES

 To provide students with relevant practical skills and experiences in their various courses

of study.

 To satisfy accreditation requirement set by National Universities Commission (NUC).

 To familiarize students with the typical work environment in which they are likely to

function professionally after graduation.

 To make transition from university to industry easy for students upon graduation and thus

enhance students’ contacts for job placement.

 To enable university educators assess their curriculum effectiveness and make imperative

amendment.

 To provide students access to equipment and other facilities that are not available in a

university laboratory or workshop and to expose them to the work methods and handling

equipment of such techniques.

 To enable students access their interests and sustainability for their chosen professions.

 To strengthen employers involvement in the whole education process of preparing

university graduates for employment in industries.

 To provide students the opportunity to see the real world of their discipline, apply their

theoretical knowledge and consequently bridge the gap between the classroom and the

real work situation.

 Students industrial work experience scheme is a program designed by the university to

expose student to the practical aspects of his/her respective course of study. It involves the

attachment of a student to an organization in line with his/her respective course of study that can

provide the training and experience required in the industry, as the experience and training

cannot be acquired in the lecture rooms but the theoretical knowledge taught in lecture rooms

shall be applied by student in real industrial situations. This training experiences, is an essential

component in the development of the practical and professional skills required of each student by

their respective course and of study and also student of biochemistry.

1.2 THE HOSPITAL

THE HISTORY OF SPECIALIST

The hospital was established in 1932 by the colonial masters, through being a general hospital

administered by several statutory authorities at respective periods of our national development

(Native Authority under the Northern Nigeria Government),Ministry of Health under the defunct

North Western State and Health Service Management Board under the former Sokoto State

Government).Between 1983 and 1989,the Hospital was utilized by the Federal government as the

Temporary Site for the Usman Dandfodio University Teaching Hospital, following which the

state took back its ownership and placed it once again under the supervision of the Health

Service Management Board. In 1992, the Hospital through an enabling edict attained its present

status of a Specialist Hospital named after the late. Head of State General Murtala Rahmat

Muhammad.
1.2.1 ORGANISATION OF THE HOSPITAL

Administrative unit- where the entire registry works is done.

Laboratory unit- which is a single unit where all the hematology, microbiology, chemical

pathology and histopathology.

Microbiology is a field in biological science that deal with the study of organisms called micro-

organisms that are too small to be seen with the naked eye except with the aid of a microscope.

Usually, an object having less than 0.1mm cannot be seen with ordinary eye, even if it can be

seen, very little or no information can be obtained.

Great scholars of microbiology postulated that; “in any case of disease, there is micro-

organism”. As a result of this, Department for Isolation and Identification of Micro-organisms

that are pathogenic using equipment and reagent for experiment was proposed

The microbiology unit was divided into different sections ranging from the Reception

section, Bacteriological bench, Parasitological bench and Serological bench which were both

explained in this report.

EQUIPMENTS USED IN MICROBIOLOGY LABORATORY

There are various equipments that can be found useful in any microbiology laboratory,

but the most vital ones include:-

Autoclave: It is used for sterilization purposes, especially prepared media before used and

certain equipments. An autoclave is made up of a closed chamber in which objects can be

subjected to steam at pressure greater than the atmosphere and therefore at temperature greater

than 1000c, and sterilization is normally achieved at 121°C for 15 minutes.

Microscope: It is an instrument that aids in observing micro-organisms and that are pathogenic,

present in sample already collected of stained or direct microscopy e.g. urine, sputum, swab,

stool and blood, etc. It also helps us to identify micro-organisms according to their

morphological structure as either bacteria gram negative, gram positive or parasites etc

Centrifuge: This is used for spinning of samples e.g to obtain serum whole.

Hot Air Oven: This is also important equipment used in the laboratory sterilization and drying

of glass wares such as Petri-dishes, slides, conical flasks etc after washing and rinsed with water

in order to make them free from microbial load.

Incubator: Micro-organisms require incubation at optimum temperature and humidity most

suited to their metabolisms and growth. Therefore, the incubator can regulate the temperature

exactly as that of the body i.e. 37°C and it is closed with constant humidity and this gives micro-
organisms ability to grow abundantly on inoculated plates. Therefore incubator is used for

isolation of microorganism.

Refrigerator: This in preservation of laboratory reagents, antibiotic test kits etc. are all stored in

the refrigerator for later use under low temperature.

Wire Loop: It is of great importance in diagnosis. It is made up of a wire which is winded and a

holder which hold the wire. It is used in culturing of microorganisms in supportive medium.

Bunsen Burner: Used for heating and sterilization purposes.

TESTS CARRIED OUT IN MICROBIOLOGY UNIT

 Urine microscope culture and sensitivity

 Stool microscope culture and sensitivity

 Sputum microscope culture and sensitivity

 Widal test

 Malaria parasite test(MP TEST)

 Acid fast bacilli(AFB TEST)

 Hepatitis test

 HIV/AIDS test

RECEPTION/COLLECTION OF SAMPLES

This is a unit which controls the collection of samples such as blood sample and urine

specimen which is forward to carry out the specify test required. The sample collection operates
between a specified times which starts by 9a.m to 3p.m. unless in case of emergency cases the

time can be altered.

SAMPLE PROCESSING

Samples are processed immediately after collection. The processing of the sample differs

from one another depending on sample and test as well.

BLOOD

A sterile, dry, preferably plastic syringe of the capacity was selected, e.g 2ml, 5ml or

10ml. A soft tubing tourniquet was applied to the upper arm of the patient to enable veins to be

felt and seen. The patients were asked to make a fist which will make the veins more prominent.

The index finger was used to feel for suitable vein, by selecting a sufficiently large straight vein

that does not roll and with a direction that can be felt. The punctured side was cleaned using 70%

ethanol and was allowed to dry. With the thumb of the left hand holding down the skin below the

puncture site, the vein puncture was made with level of the needle directed upwards in the line of

the vein to fill. Moving the needle in to the vein was avoided.

When sufficient blood has been collected, the tourniquet was released and the patient was

instructed to open his/her fist. The needle was removed and immediately the punctured site was

pressed with a piece of dry cotton wool. The tourniquet was removed completely. The patient

was instructed to continue pressing on the punctured site until the bleeding stops. The needle was

removed from the syringe and was discarded safely, and the blood was mixed immediately in

sodium citrate test tubes, depending on the type of test requested.

URINE
Urine specimen is collected by given the patient a urine sterile container and shows the patient

on where to go and ease. Sufficient urine is required.

After collection, the urine is observed physically, chemically by dipping a urine combi strip for

observing so particular substances or metabolites in the urine.

The urine is spin using a centrifuge so to get precipitate by discarding the precipitate. A small
portion of precipitate is drop on a free grease slide for microscopy

MATERIALS USED IN THE RECEPTION

 Slides and cover slip: this is a transparent rectangular glass used for viewing blood

sample after smears for malaria parasite, reticulocyte count wet preparation under high

power microscope

 Syringe: This is used for collection of blood samples from patients intravenously. It be

could be 2ml, 5ml and 10ml.

 Tourniquet: use to tighten the arm of the patient to make the veins more visible and to

stop the flow of blood through an artery when collecting blood samples from the patient.

 Hand gloves: used to prevent the hands from touching blood samples when the various

test are being carried out. This is usually used in the laboratory for handling samples to

prevent infections from been transferred from the sample to the person carrying out the

test.

 White tile: used to see for the agglutination reaction in the blood samples when testing

for its blood group.

 The cotton wool: This is a whitish-soft material, used for sterilization when dipped into

the methyl ethanol. Also used for general cleanings in the laboratory.

COMMON LABORATORY REAGENT

Crystal Violet Dye: This is made up of crystal violet powder, ethanol, ammonium and distilled

water and it’s a basic dye. Crystal violet is used as primary stain in gram staining which gives the

gram positive organism a dark purple color which resist decolonized by alcohol or acetone.

Followed by Gram Iodine or Lugol’s Iodine, Acetone, then lastly Safranin which is used as

the secondary dye in gram staining.

Acetone (Alcohol): Acetone-alcohol is made by mixing distilled water with ethanol and then

acetone is added into the solution and mixed well. This solution acts as decolonizer of primary

stain in gram staining by making those organism that are not gram positive to lose the colour of

primary stain (dark purple) and assume that of the counter stain (pinkish or red).

Lugol’s Iodine: This consists of iodine and potassium iodide, using a clean brown bottle,

potassium iodide is first dissolved in distilled water followed by the addition of iodine. The

solution is stored in a dark place at room temperature. This reagent acts as mordant (i.e. it serves

as a linker between the primary stain crystal violet) and the tissue of the organism.

Stained Salmonella Antigen Kit: Industrially prepared antigen kit for Salmonella typhi,

Salmonella paratyphi A, Salmonella paratyphi B, and salmonella paratyphi C for both their

somatic “O” and flagella “O” antigen which are the causative agent of typhoid fever are

available in a good and preserved temperature.

MEDIA PREPARATION

Each media is prepared as directed by the manufacturers in other to be supportive. For example,

28g of nutrient agar powder directed to be dissolve in 1L to obtain a normal concentration.

28g ———— 1000ml/1L

And therefore each Petri-dish contains approximately 20 ml of prepared medium. If one in need

to prepared less than 1L quantity, certain calculations need to be done in order to maintain a

normal concentration. E.g.

To prepare 10 plates volume of a medium which is 200ml, the following formula has to be

followed:

28g ———— 1000ml/1L

But x ———— 200ml

Cross multiply

x=28 × 200 = 5.6g

1000

Therefore 5.6g is to be weighed and dissolve in 200ml of distilled water to have same

concentration as if you are dissolving 28g in 1000mls of water. Nutrient Agar being the basic

media in the formation of some enrichment media e.g. Blood and Chocolate Agar, it is also used

in conducting sensitivity test through arranging antibiotic disc on it.

Nutrient Agar Preparation

Materials: Weighing balance, non-absorbent paper, conical flask, autoclave, Bunsen burner,

Petri-dishes, powdered media, cotton wool, distilled water, aluminium foil, spatula, measuring

cylinder, .
Method: required amount of powder media (5.6g) will be dissolve in required volume (200ml)

of distilled water as the requirement from the above calculation, in a conical flask. The mouth of

the conical flask will be close with cotton wool, wrap with aluminium foil, and the mixture will

be shake vigorously and heated on burner flame to mix completely, and then sterilize the

medium by autoclaving at the temperature of 121°C for 15 minutes. After sterilization the

prepared will be allow to cool to about 50°C then dispense into sterile Petri-dishes to solidify. If

few bubbles appear on the surface of freely poured plate, they are removed quickly by passing

the flame from the Bunsen burner over the surface of the dispersed media.

The media will then be allow to gel before been taking to fridge for preservation.

Blood Agar Media: This is made by incorporating nutrient agar with blood. Most bacteria of

medical importance grow well on it such as Streptococcus pneumoniae and Neisseria spp it is

used in sensitivity test and also in detecting haemolytic bacteria.

Preparation: After preparing the nutrient agar as mentioned earlier, It was then allowed to cool

before it gel, then blood is added and mix gently, then blood agar is then poured in sterile Petri-

dishes.

Chocolate Agar: This agar serves as differential media. It is prepared through lysing of the red

cells in the blood.

Preparation: After preparation of blood agar media, it is heated till the media turn

brown/chocolate in colour, allowed to cool to 40°C, then dispense into separate Petri-dishes

Mac Conkey Agar Media: This media acts as a selective and differential media. It is used for

isolation of intestinal bacteria e.g. Escherichia coli, klebsiella spp e.tc.

Preparation: it’s prepared the same with Nutrient agar.

CLED agar (Cystine lactose electrolyte deficient): Medium use for the cultivation of gram-

postitive and gram-negative urinary tract bacteria. CLED Agar is a non-selective differential

plating medium for the growth and enumeration of urinary tract microorganisms. Omitting

sodium chloride CLED Agar inhibits the swarming of Proteus.

Preparation: it’s prepared the same way like others; nutrient agar and Mac conkey agar.

CHAPTER TWO

2.0. BACTERIOLOGY SECTION

This section is one of the sections in microbiology department which deals with the study of

bacteria. Bacteria are prokaryotic organisms with different shapes which are classified into

sperical (cocci), rod-shaped (bacilli), Spirocate (spiral) and comma-shaped (vibro).

The most common diagnostic methods applied in bacteriology laboratory include

staining, microscopy and culture, and sensitivity test. The specimens used in detecting bacterial

pathogens include urine, sputum, swabs, etc.

Some common diagnostic methods applied in bacteriology section are explained below:-

2.1. GRAM STAINING

 This is a technique that differentiates bacteria into two major groups, Gram positive and

Gram negative bacteria based on their cell wall composition, this technique was produced by Sir

Christian Gram in 1883.

Principle: Gram positive bacteria is known to have large amount of peptidoglycan and small

pore sizes, it therefore resist decolarization by alcohol and take the color of primary dye

(blue/purple), where by Gram negative bacteria has a small amount of peptidogly and large pore

sizes, which can be affected by decolarization of alcohol, so its take the color of secondary dye

and appear red in color.

Materials and Reagents: Wire loop, Bunsen burner, glass slide, crystal violet (primary dye),

gram iodine, alcohol, neutral red (secondary dye), tap water, emersion oil and microscope.

Specimen: An overnight cultured organism

Method/Procedure: Place a drop of distilled water on a centre of a clean grease free glass slide

with the aid of pasture pipette, sterilize your wireloop in a burner flame until it become red to

hot, allow it to cool freely, pick a colony of test isolates and mix with drop of distilled water on a

slide, spread the mixture to make a smear of 1cm in diametre. Allow the smear to air dry, pass it

on burner flame 3-4 times to fix, then take it to a staining rack for gram staining.

After the smear is prepared, it undergoes the gram staining as follows:-

 Place your slides on a staining rack.

 Cover the smear with crystal violet and allow standing for 1-2mins, and then washing

gently with running tap water.

 Cover the smear with lugol iodine for 30-60 seconds.

  Flood the smear briefly with acetone/95% methylated spirit (decolorizer) until no stain

runs, rinse the slide with water

 Cover the smear with safranin or neutral red for 1mins, wash with water.

 Wipe the back of the slide with cotton wool, and place the slide on draining rack to air

dry (at room temperature).

 Place a drop of emersion oil on stained smear and examine under oil immersion lens

(×100) objective.

Observations:- Gram positive bacteria take the color of primary dye and appear blue or purple,

while Gram negative bacteria take color of secondary dye and appear red or pink.

2.2. CULTURE OF MICRO-ORGANISMS

Materials: Sample (e.g. urine), wireloop, burner flame, prepared medium (e.g. CLED agar),

incubator.

Procedure: Sterilize your wireloop until it become red hot, allow it to cool with free air, dip the

wireloop in a sample (urine) to pick an inoculum, transfer in to a prepared medium (CLED agar),

streak accordingly to obtain primary, secondary and tertiary inoculation, then take the cultured

plate to an incubator for 24h at 37°C.

Observation: After 24 h of incubation, the cultured plates are going to be observed the presence

of colonies that might grow due bacterial infection that comes from the sample. Different
colonies ranging from lactose fermenting bacteria such as Escheriachia coli, Klebsiella sp,

Enterococcus feacalis to non-lactose fermenting bacteria such as: Pseudomonas aeruginosa,

Proteus vulgaris and mirabilis and late lactose fermenting bacteria such as: Citrobacter sp, with

different colony sizes, edge shape, odor, e.t.c. on the cultured plates.

2.3 SENSITIVITY TEST

This is a test that is carried out to know the actual antibiotics that is susceptible to the isolated

bacteria, and those that are resistant by the bacteria isolated from patient sample. This can be

done by sub-culturing the identified isolate (microorganism) into another fresh non inhibitory

medium and spread all over the plate, then antibiotics will be inserted after then incubate the

plates in an incubator for 24h at 37°C.

Observation: Zone of inhibition around each antibiotic is going to be observed which determine

its susceptibility; those that create no zone of inhibition are the resistant antibiotics

CHAPTER THREE

3.0 PARASITOLOGY SECTION

Parasitology is a branch of science that deals with the study of parasites. e.g. Entamoeba

histolytica, Ascaris lumricoides, Giardia lamblia, Trichuris trichuria,Schistosoma

haematobium,Trichormonas vaginalis etc. Investigations of parasitic infection in microbiology

laboratory includes: Urine microscopy, stool microscopy, and malaria parasite diagnosis.

Microscopy: Is an observation, investigation or experiment that involves the use of microscope.

There are two types which are:

I. Direct microscopy or wet preparation

 II. Non-direct microscopy; which staining takes place before microscopy

Direct Microscopy: mostly urine, stool and semen undergo this process by viewing the

specimen directly.

Microscopy which is not direct: This required staining of specimens before viewing under

microscope, mostly are sputum for AFB, gram staining, e.t.c.

3.1 URINE MICROSCOPY

Material: Centrifuge, test tube, glass slide, cover slip, microscope, sample.

Procedure:

 pour 5-10ml of urine sample in a test tube

 Spin the sample using centrifuge at a speed of 3-5000rpm for 5 mins,

 Discard the supernatant layer by completely pouring out the urine, while leaving the

sediment that remain at the bottom of the test tube

 Place a drop from the sediment on a clean glass slide, and cover with cover slip, avoid air

bubbles.

 View under ×10 and confirm with ×40 objective lens.

Observation

In urine microscopy, things that could be seen in includes: pus cells, red blood cell, epithelial

cells, yeast cells, casts (hyline, waxy, cellular, granular), crystals (calcium oxalate, triple

phosphate, tyrosine, sulphonamide, cystein, cholesterol), parasites (Schistosoma haematobium,

Trichomonas vaginalis, Onchocerca vulvulus).e.t.c.

3.2 STOOL MICROSCOPY

Materials: slide, cover slip, applicator stick, malachite green/iodine, stool sample, microscope,

normal saline, pipette.

Procedure:

 pipette few drops of normal saline on a slide,

 Use applicator stick and pick a small amount of stool sample,

 Mix with normal saline on the slide,

 Remove coarse particles, add drop of malachite green,

 Mix the preparation homogeneously,

 Cover the preparation with cover slip,

 View under ×10 and ×40 objectives.

Observation: parasites; such as Entamoeba histolytica, Giardia lamblia, Ascaris lumbricoides,

e.t.c. could be seen.

3.3 MALARIA PARASITE TEST

Malaria is a disease caused by a protozoan parasite of the genus Plasmodium. It has five

causative agents: P. falciparum, P. ovale, P. malariae, and P. vivax. Malaria diseases in human,

is transmitted throught the bite of female anoples mosquito. This parasite can be detected in the

lab through:

 Microscopy of stained blood film

  Use of Rapid Diagnostic Test kit.

The causative agent of this disease is plasmodium which is acquired via mosquito bite. Test is

carried out in the laboratory to detect the parasite in the blood sample of patient and to identify

the stages of development of parasites.

Microscopy of stained slide: there are two methods: thick blood film, and thin blood film.

Thick film procedure

 Drop a fresh blood on a centre of clean free grease glass slide

 Use a spreader and wide the blood to make a thick film of 1cm in diameter

 Allow the blood to air dry

 Stained the blood film with geimsa stain for 45 minutes.

 Rinse the film with distilled water and air dry

 Put a drop of immersion oil on stained film

 View under ×100 lens

Thin film procedure

 Drop a fresh blood at the edge of a clean grease free glass slide

 Use a spreader and touch the drop of blood by the edge

 Make a forward move with touched blood to obtain a thin film with head, body and tail

shape

 Allow the film to air dry

 Flood the film with methanol to fix for 10 secs

 Stained with geimsa reagent for 45 mins

  Rinse and air dry on draining rack

 View the stained film under microscope with ×100 objective

 Report the presence of malaria parasite

Observation: A circular shaped structure with red chromatin dots is trophozoite of Plasmodium

spp. Malaria parasite is recognized when stained with giemsa reagent by its red staining nuclear

materials and blue staining cytoplasm and by its occurrence within the red blood cells (RBC).

CHAPTER FOUR

4.0 SEROLOGY SECTION

Serology can be defined as the branch of medicine concerned with study of blood serum

and its constituents especially its role in protecting the human body against diseases. However,

this chapter will precisely discuss test such as widal test, Hepatitis and HIV screening.

4.1 WIDAL AGGLUTINATION TEST

This serological test involved Antigen-Antibody interaction with form agglutination. It is

carried out in the laboratory to detect the presence of Salmonella spp in the blood of an infected

individual. Serum is used in carrying out the test, together with prepared stained salmonella
antigen kit. The reagent in the kit included somatic Antigen (O) and flagella Antigen (H), both

the O and H type has their paratyphi (sub-species) A, B and C

Materials: Test-tubes, white tile, centrifuge, prepared stained salmonella antigen kit, disposable

stirrers and pasture pipettes.

Procedure:

 The blood collected from patient is poured in a clean test tube

 Spin the blood sample in a centrifuge at the speed of 3-5000rpm for 5 minutes

 The serum that settled top in the tube is pipette and drop on clean white tile into 8 drops

points

 Each stained Salmonella antigen is drop on serum respectively

 Disposable stirrer is use to mix the serum and the antigen kit drops

 The tile is rock for about 1-2 minutes

 Agglutination is observed and report

Observation

Appearance of agglutination is as a result of antigen-antibody reaction which indicates typoid

fever (Salmonella) infection. Diagnostic titre starts from 1/80 and above.
4.2 HEPATITIS TEST

Viral hepatitis is a systemic primarily involving the liver. Most cases of acute viral hepatitis are

caused by Hepatitis A virus, Hepatitis B (HBV), or Hepatitis C virus. One step Hepatitis antigen

test strip (serum/plasma) is a rapid test to qualitatively detect the presence of Hepatitis in serum

or plasma specimen. The test utilizes a combination of monoclonal and polyclonal antibodies to

selectively detect elevated levels of Hepatitis in serum or plasma.

Principle: The membrane is pre-coated with anti-hepatitis antibodies on the test line region of

the test strip. During the testing, the serum or plasma specimen reacts with the particle coated

with anti hepatitis antibody. The mixture migrates upward on the membrane chromatographically

by capillary action to react with anti-hepatitis antibodies on the membrane and generate a colored

line. The presence of this colored line indicates a positive result, while its absence indicates a

negative result. To serve as a procedural control, a colored line will always appear in the control

line region indicating that proper volume of specimen has been added and the membrane wicking

has occurred.

Materials: Hepatitis A, B, or C test strip, specimen collection container, centrifuge, timer.

Procedure:

 Bring the pouch to the room temperature before opening it.

 Remove the test strip from the sealed touch and use it as soon as possible. Best result will

be obtained if the assay is performed within one hour.

 With arrows pointing towards the serum or plasma specimen, immerse the strip vertically

in the serum or plasma for at least 10-15 seconds. Do not pass the maximum line (MAX)

on the test strip when immersing the strip

 Place the test strip on a non-absorbent flat surface

 Start the timer and wait for the red line(s) to appear. The result should be read at 15

minutes.

NB: a low Hepatitis concentration might result in a week line appearing in the test region (T)

after an extended period of time; therefore, do not interpret the result after 30 minutes.

Observation: Positive result shows two distinct red lines appear (one should be in the control

region (C) and another line should be in the test region (T). Negative result shows one red line

appears in the control region (C). No apparent red or pink line appears in the test region (C).

Invalid result shows no control line; the reason is that in sufficient specimen volume or in correct

procedural techniques are the most likely reasons for control line failure. Therefore review the

procedure and repeat the test with a new test strip again. If the problem persists, discontinue

using the test kit immediately and contact your local distributor (supervisor).
4.3 HIV SCREENING

Human Immunodeficiency Virus (HIV) has been recognized as the etiological agent of the

acquired immunodeficiency syndrome (AIDS). Several types of rapid test kits use in screening of

HIV include: Determine strip, Uni-Gold test kit, Start pak test kit e.t.c. Test using these kits is

based on the immune chromatographic sandwich principle.

Principle: During testing two drops of serum, plasma or whole blood is applied to the sample

port, followed by two drops of wash buffer and allowed to react. Antibodies of any

immunoglobulin class specific to the recombinant HIV-1 or HIV-2 proteins will react with the

colloidal gold linked antigens. The antibody protein colloidal gold complex moves

chromatographically along the membrane to the test and control regions of the test device. A

positive reaction is visualized by a pink/red band in the test region of the device. A negative

reaction occurs in the absence of human immunoglobulin antibodies to HIV in the analysed

specimen. Consequently no visually detectable band develops in the test region of the device.

Excess conjugate forms a second pink/red band in the control region of the device. The

appearance of this band indicates proper performance of the reagents in the kit.
Materials: Timer, Test kit device, wash buffer, Disposable pipettes, test tube (container),

capillary tubes, lancet.

Procedure:

 If any reagent/sample has been in refrigerated storage, remove and allow to stand to reach

for at least 20 minutes to reach room temperature

 Remove the required number of test device from their protective wrappers.

 Label each test with the appropriate patient information.

 Using one of the disposable pipettes supplied, fill with sample (serum, plasma or whole

blood).

 Holding the pipette over the sample port add two drops of sample (approximately 60µl)

carefully.

 Add 2 drops (approximately) of wash reagent/buffer to the sample port.

 Allow 10 minutes from the time of wash buffer/reagent addition for reaction to occur.

The result should be read immediately after the end of the 10 minutes after the incubation

time. Do not read result after 20 minutes following sample addition.

Observation: A line in the control region only indicates a negative result. A line of any

intensity forming in the test region, plus a line forming in the control region indicates a positive

result. If no line appears in the control region, the test should be repeated with the fresh device

irrespective of a line developing in the test region.

NB: failure to allow kits to come to room temperature prior to use may impact results.

4.4 BLOOD GROUPING

Method: Tile method

Aim: to determine the blood group

Introduction: In 1901, Karl Landsteiner discovered that human blood could be grouped into

principal types, which were designated as A,B,AB, and O this method of classification is called

ABO blood group system when blood between two individual is mixed, agglutination occurs.

This clumping is because of immunological reactions. This reaction is due to antigen antibody

reaction. Landsteiner found two antigens in RBCs and named as A antigen and B Antigens. He

noticed the corresponding antibodies in the serum called anti A and B. however in the body, a

particular antigen and the corresponding antibody cannot be present together, if present if causes

clumping of the blood. Based on the presence or absence of antigen A and B blood is divide into

four group A,B,AB, and O.

Requisite for blood typing. Antiserum A and antiserum B tile

Principle: The blood grouping is done on the basis of agglutination. Agglutination means the

collection of separate particles like RBCs into clumps or masses agglutination occurs if an

antigen is mixed with its corresponding antibody called agglutination occurs if antigen A is

mixed with anti A and vice versa

Procedure

Antisera A Antisera B Antisera D BLOOD TYPE

A+

B+
 AB+

O+

Key

Agglutination reaction occur

No agglutination occur

 One drop antiserum A is place on one end of the tile and one drop antiserum B at the middle

and antiserum D on the other end

 One drop of red blood suspensions is mixed with each antiserum. The tile is slightly rocked

for two minutes. The presence or absence of agglutination is observed

Absence of agglutination is confirmed by clear mixture with dispersed RBCs

Result: If agglutination occurs antiserum A, so the blood group is A

If agglutination with antiserum B, so the blood group is B

If agglutination occurs antiserum both antiserum, then it is AB

If agglutination occurs antiserum does not occur with either of the antiserum, the blood group is

O Importance of ABO blood in blood transfusion during blood transfusion, only the compatible

blood must be used. The one who gives blood is called the donor and who receives is called

recipient while blood transfusion, antigen of the donor and the antibody of the recipient are

considered. The antibody of the donor is ignored mostly. The RBC of O group has no antigen

and so agglutination does not occur with any other group of blood. So O group blood can be
given to any blood group persons hence the name universal donor. The plasma of AB group

blood has no antibody. This does not cause agglutination of RBC from any other group of blood.

Thus blood group is a universal recipient

4.5 PACKED CELL VOLUME (PCV) TEST

Introduction: The haematocrit (Ht or HCT, British English spelling haematocrit), also known as

packed cell volume (PCV) or erythrocyte volume fraction(EVF), is the volume percentage (%) of

red blood cells in blood . It is normally 45% for men and40% for women. It is considered an

integral part of a person's complete blood count results, along with haemoglobin concentration,

white blood cell count, and platelet count. Because the purpose of red blood cells is to transfer

oxygen from the lungs to body tissues, a blood sample's haematocrit—the red blood cell volume

percentage can become a point of reference of its capability of delivering oxygen. Additionally,

the measure of a subject's blood sample's haematocrit levels may expose possible diseases in the

subject. Anaemia refers to an abnormally low haematocrit, as opposed to polycythaemia, which

refers to an abnormally high haematocrit. For a condition such as anaemia that goes unnoticed,

one way it can be diagnosed is by measuring the haematocrit levels in the blood. Both are

potentially life-threatening disorders.

Principle: The packed cell volume is that proportion of whole blood occupied by RBCs when it

is packed together, expressed as a ratio (litre/litre).Anticoagulated blood in a glass capillary of

specified length, bore size, and wall-thickness is centrifuged in a micro haematocrit centrifuge at

RCF 12 000–15 000 xg for 3–5 minutes to obtain constant packing of the red cells. A small

amount of plasma remains trapped between the packed red cells.

The PCV value is read from the scale of a micro haematocrit reader (we will see it later) or

calculated by dividing the height of the red cell column by the height of the total column of

blood.

Note: Due to trapped plasma, PCV values using a centrifugation technique are 1–3% higher than

those obtained when using an electronic cell analyser which computes the value from the MCV

and red cell count.

Material/Reagent: Micro haematocrit centrifuge Scale reader (usually provided with the

centrifuge) Capillary tubes, 75mm long with a 1.5-mm bore, containing dried heparin (if

capillary blood is used; if venous blood mixed with EDTA dipotassium salt, 10% solution

(reagent no. 22) is used, “heparinized” tubes are not required) Filter-paper Soft wax or plastic

modelling clay (or a Bunsen burner or spirit lamp) Sterile blood lancet70% Ethanol. Micro

haematocrit centrifuge the main 2 parts in the micro haematocrit centrifuge are:

1. The Haematocrit Rotor: which held the capillary tubes in micro haematocrit centrifuge:

– The places that we put the capillary tubes in are numbered and you should write down

the number of each tube on the cheat of the corresponding sample to avoid writing the

wrong result in the wrong sample cheat.

2. The haematocrit reader: It is the scalar by which we measure the percentage of RBCs

column to the total column. – It measures the percentage of the fraction directly not the

length of the columns– There are 3 major types of scalar; haematocrit reader card, spiral

micro-haematocrit reader and the circular micro-haematocrit reader but we will talk about

how to use each one in the procedure.

Collection of specimen: Collection of specimens Capillary blood specimens using a blood

lancet, draw blood by pricking either:

  The third or fourth finger

 The lobe of the ear

 The heel (infants)

After sterilizing the chosen area with ethanol, the blood should flow freely or with very little

pressure to the area. Wipe away the first drop with filter-paper.

Plugging or sealing the capillary tube: Plug the end of the tube that has not come into contact

with the blood with soft wax or plastic modelling clay.

  Check that it is completely plugged to a depth of about 2mm Clay. Alternatively, seal the

end of the tube by heating it carefully over a Bunsen burner or spirit lamp.

 Leave it to cool in a horizontal position. Sealing the capillary tube by the flame from a

spirit lamp or pilot flame from a Bunsen burner can distort the glass, causing breakage

when the internal lid is screwed down on the rotor. Red cells may also be lyzed by the

heated glass. Use of an open flame is also a fire hazard. It is useful to have ready a

numbered stand containing plastic modelling clay, so that each patient’s tube can be stuck

upright next to the corresponding number

Measurement technique:

Centrifugation:

1. Place the capillary tubes in the numbered slots in the centrifuge head. – Making sure that the

number on the slot corresponds to the specimen number. – The sealed end of the tube should

point outwards, away from the centre to avoid blood leaking out during centrifugation– Make

sure that you balanced the micro centrifuge by putting another capillary tube opposite to that

of the sample. Micro centrifuge balance the clay end outside away from the centre

2. Centrifuge at 3000 g (for the period of time recommended by the manufacturer of the

centrifuge — usually 10 minutes).After centrifugation, the tubes will show three layers:— at

the top, a column of plasma;— in the middle, a very thin layer of leukocyte sand platelets ;—

at the bottom, a column of erythrocytes. Now we get ready to read the results on the

haematocrit reader.

Reading the result on the hematocrit reader

The erythrocyte volume fraction (i.e. reading the PCV) reading is made exactly at the top of the

Column of erythrocytes using the haematocrit card and advanced reader card.
There are many types of the manufactured reader cad but we will get one type for example Hold

the tube against the scale so that the bottom of the column of erythrocytes (not the bottom of the

tube) is aligned with the horizontal zero line. Move the tube across the scale until the line marked

1.0 passes through the top of the plasma column. Check to make sure that the bottom of the

column of erythrocytes is still on the zero line; also check (by means of the heavy vertical lines)

that the tube is vertical. Move the reader bar until its line passes through the top of the RBCs

column.

Aims: The packed cell volume (PCV), also referred to as haematocrit is used to screen for

anaemia when it is not possible to measure haemoglobin accurately and mains electricity is

available to operate a micro haematocrit centrifuge (you will see it soon).Value of PCV (i.e. the

RBCs region) ==> The PCV is also used in the investigation of dehydration, burns, and

polycythaemia (i.e. a condition in which the plasma volume is decreased or the RBCs number

increased).

Microhematocrit reader

4.6 PREGNANCY TEST (PT)

The objective of this test to detect the presence of human chorionic gonadotropin (hcg) in

women.
APPRATUS: Centrifuge, PT strip, serum or urine specimen.

PRINCIPLE: HCG is a hormone secreted in to body by oestrogen through the stimulation of the

pituitary gland in pregnancy and is presence in plasma, serum and urine of pregnant woman. IT

can be detected through antigen antibody reaction.

PROCEDURE: The blood or urine will collected, for blood it will be centrifuge for 5 minutes.

After separation the strip was dipped in to the plasma or urine specimen and allowed to touches

the margin line and will read after 20 minutes.

Pt strip

TEST T T

CONTROL C C

SAMPLE REGION

Negative Positive Invalid

RESULT: A double line indicate positive result (test and control line) while single line indicate

negative result (control line), when test line only appear, it indicate invalid result.
4.7 SYPHILIS TEST (VDRL).

Syphilis is a sexually transmitted infection caused by the spirochete bacterium

Treponemapallidum subspecies pallidum. The primary route of transmission is through sexual

contact; it may also be transmitted from mother to foetus during pregnancy or at birth, resulting

in congenital syphilis. Other human diseases caused by related Treponemapallidum include yaws

(subspecies pertenue), pinta (subspecies carateum), and bejel (subspecies endemicum).The signs

and symptoms of syphilis vary depending in which of the four stages it presents (primary,

secondary, latent, and tertiary). The primary stage classically presents with a single chancre (a

firm, painless, non-itchy skin ulceration), secondary syphilis with a diffuse rash which frequently

involves the palms of the hands and soles of the feet, latent syphilis with little to no symptoms,

and tertiary syphilis with gummas ,neurological, or cardiac symptoms. It has, however, been

known as “the great imitator" due to its frequent atypical presentations. Diagnosis is usually

made by using blood tests; however, the bacteria can also be detected using dark field

microscopy. Syphilis can be effectively treated with antibiotics, specifically the preferred

intramuscular benzathine penicillin G (or penicillin G potassium given intravenously for neuron

syphilis), or else ceftriaxone, and in those who have a severe penicillin allergy, oral doxycycline

or azithromycin

Procedure

1. Obtain 2-3 ml of blood from the patient using standard vein puncture technique. Collect

the Blood into an anticoagulant free (i.e. "clot") tube or serum separator tube (SST).

Plasma (purple Top tube) may be used up to 48 hrs after collection.

2. Label tube with patient's name and date of collection.

 3. Complete the Test Requisition Form or order request online through our Laboratory

Information Management System (LIMS).Specimen and forms should be placed in a

designated location at each site for the lab courier to pick up.

4. Allow specimen to clot and label with a unique identifier to match requisition form.

Wherever a Centrifuge is available, specimens should be spun on the same day of

collection. Arrange for Transport to laboratory. If transportation is delayed, refrigerate

tube to minimize haemolysis.

Aims: To detect syphilis (sexually transmitted disease) in a patients, usually females

Materials/Reagents

 Blood sample,

 serum, EDTA bottle

 syringe and needle

 syphilis strip

 Swap

 cotton wool

 Micropipette

 Table centrifuge

Result: If your test comes back negative for syphilis antibodies, the result suggests that you

don’t have syphilis. If your test comes back positive for syphilis antibodies, you probably (but

not definitely) have syphilis. If this occurs, your doctor will order a more specific test to confirm

the Results. A treponemal test is often used to confirm the positive test. Treponemal tests check

whether your immune system has produced specific antibodies in direct response to the syphilis-

causing Treponemapallidum.
Risk: The risks of a blood draw are fairly minor. You might have slight issues like mild pain

during the Blood draw or minor bruising or bleeding afterward. Developing a serious problem

from a blood draw, such as inflammation of the vein or an infection, is rare.