CHAPTER TWO

2.1. GENERAL LABORATORY EQUIPMENTS

The Light Microscope
The microscope employs a hollow, extremely intense cone of light concentrated on the
specimen. The field of view of the objective lens lies in the hollow, dark portion of the cone and
picks up only scattered light from the object. The clear portions of the specimen appear as a
dark background, and the minute objects under study glow brightly against the dark field. This
form of illumination is useful for transparent, unstained biological material and for minute
objects that cannot be seen in normal illumination under the microscope.
Autoclave
The autoclave is effective equipment used for steam sterilization at pressures above the
atmospheric pressure. Thus, it is possible to steam at higher temperature then the boiling point,
which a lot of microorganisms cannot withstand. Autoclaving is the most effective method for
sterilizing culture media. When sterilizing culture media with autoclave, we do so at 1.05Kg per
square centimeter for 15 minutes to eliminate contaminations.
Refrigerator
This is used to preserve samples, reagents etc, which are used for daily analysis and cannot be
exhausted at once. The refrigerator helps provide optimum environment for materials to be
preserved. Incubator
The incubator is mainly used to incubate culture media as microbes have different optimum
temperatures for growth and reproduction. The temperature of an incubator can be set to
the preferred temperatures.
Water Bath
This is required to incubate bottle of culture media, liquids in flasks or other large
Containers, and when incubating samples in the test tube racks.
Weighing Balance
This is a delicate instrument used for weighing essential, reagent, stains and culture
media that requires adequate weighing.
Straight Wire
It is made up of a thick metallic lower part and a straight thin upper metallic part usually made up of
platinum. This straight wire is used for stab culture and for picking discrete colonies. Usually
sterilized before, during and after usage. This is achieved by flaming on Bunsen burner red hot and
allowed to cool a bit before use.
Wire Loop
Made up of a thick metallic lower part and a straight thin upper metallic part curved into a small circle
usually made up of platinum. Wire loop is used generally for inoculating samples and picking
colonies sterilized by flaming red hot before, during and after use. It is always better to use the sides
of the loop rather than the apex during inoculation.
Mycology Needle
It is made up of a thick metallic lower part and a short straight thin upper metallic part usually made
up of platinum. Used for needle mount preparations of fungi and fungi inoculation. It is usually
sterilized by flaming.
Glass Slides
Used for preparation of slides for microscopy. Sterilization is by flooding with alcohol and flaming
off excess alcohol.
Cover Slips
This is use for covering wet smears of preparations. It is sterilized by flooding with alcohol and
flaming off excess alcohol.
Petri Dish
Used for the preparation of culture media. It is usually bought sterilized. The disposable type cannot
used a second time while the glass ware type can be reused be usually sterilized by autoclaving.
Forceps
A pair of forceps is a metallic object used for handing hot object or contaminated materials. It is
sterilized by flaming red hot.
Others include:
Sterilized slide, Giemsa Stain, needle, syringe, ethanol, sterilized bottle, agar (MacConkey or
Chocolate), Gram positive, Gram negative sensitivity kit , cotton wool, EDTA, microscope, oil
immersion, ethanol, sterilized slides, swab sticks, cotton wool, spirit, giemsa stain, lancets,
surgical blades, oil immersion, pipette, light microscope, hot plate (dryer), centrifuge, hand
gloves, microhaematocrit centrifuge, capillary tubes for measuring PCV, sealant,
microhaematocrit reader, anaerobic jar, test tubes, bottles, water bath, weighing balance,
microscope, pipette, beakers, bio safety cabinet, cotton.

2.2. SAFE WORKING PRACTICES IN A MEDICAL LABORATORY.

The following are some of the important points which apply when working with infectious
materials:
1. Never mouth-pipette. Use safe measuring and dispensing devices.
2. Do not eat, drink, smoke, store food, or apply cosmetics in the working area of the
laboratory.
3. Use an aseptic technique when handling specimens and cultures.
4. Always wash your hands after handling an infectious material in the laboratory, when
leaving the laboratory and before attending to patients. Cover any open wound with a
water proof dressing.
5. Wear appropriate protective clothing when working in the laboratory. Ensure it is
decontaminated and laundered correctly.
6. Wear protective gloves and when indicated a face mask, for all procedures involving
direct contact with infectious materials. When wearing gloves, the hands should be
washed with the gloves on, particularly before doing ant clerical work.
7. Centrifuge safely to avoid creating aerosols. Know what to do should a breakage
occur when centrifuging.
8. Avoid practices which could result in needle stick injury.
9. Do not use chipped or cracked glassware and always deal with a breakage
immediately and safely.
10. Avoid spillages by using racks to hold containers, work neatly and keep the bench
surface free of any unnecessary materials.
11. Decontaminate working surfaces at the end of each day’s work and following any
spillage of any infectious fluid.
12. Report to the laboratory officer in charge, any spillage or other accident involving
exposure to infectious material.
13. Know how to decontaminate specimens and other infectious materials.
14. Use and control an autoclave correctly.
15. Dispose laboratory waste safely.
 CHAPTER THREE

3.1. THE RECEPTION

The receptionist on seat, collects samples from patients waiting to be transferred to the
laboratory, put bills on the patients cards depending on the kind of tests to be done, register the
patients cards and then also register results before they are given out to patient, they also give out
universal, anticoagulant bottles to patient and give them necessary instructions on how to collect
into the bottles that is being given to them. Some of the laboratory materials are stored in the
reception. Listed below are a few steps to follow when dispatching microbiological specimens:
1. Keep a register of all specimens dispatched. Record the name, number, and ward or
health centre of the patient, type of specimen, investigation required, date of dispatch,
and the method of sending the specimen. When the report is received back from the
microbiology laboratory, record the date of the receipt on the register.
2. Check the specimen container is free from cracks, and the cap is leak-proof.
3. Use sufficient packaging material to protect a specimen especially when the container is a
glass tube. When the specimen is fluid use sufficient absorbent material to absorb it
should a leakage or breakage occur.
4. Mark all specimens that may contain highly infectious organisms.

3.2. MICROBIOLOGY LABORATORY

In this laboratory the following tests are carried out test
• Urine Analysis, MCS
• Malaria Parasite Test
• Stool microscopy
• Semen Analysis
• Blood Microfilaria

3.2.1. URINE ANALYSIS, MCS.

URINE BENCH
Pathogens that could be found
Bacteria
• Gram positive: Staphylococcus, Hemolytic Streptococci
• Gram negative: Escherichia coli, Proteus species, Pseudomonas, Aeruginosa, Klebsiella
strains, Salmonella typhi, Neisseria gonorrhea.
The following activities are carried out on the urine bench:
Urine macroscopy i.e. Appearance which includes the color, turbidity etc. The microscopy to
check out for possible parasite. Then culture of urine samples.
URINE MACROSCOPY AND MICROSCOPY
Some other urine parasites include Wucherariabrancoftii, Onchocerca etc.
Collection of urine
Urine is collected in clean universal bottles. The mid part of the first early morning sample is
preferred.
MACROSCOPIC EXAMINATION
Appearance: the normal urine color should be either amber or yellow. Other colors could be red
brown black or white.
Turbidity: it could be slightly turbid, turbid or clear.
MICROSCOPIC EXAMINATION
Note: You culture before you spin the urine samples in the centrifuge to avoid contamination the
samples.
The urine samples are poured inside test tubes and labeled with the laboratory number of
the patient. It is then arranged inside the centrifuge and allowed to spin for 10minutes, so as
to separate the urine into layers.
The supernatant part of the spinned urine is then disposed off into a container containing a
disinfectant and then the sediment is placed on the glass slide. The sediment of urine sample on
the slide is covered with a cover slip and then examined under the microscope. The following
could be seen under the microscope: bacteria cells, epithelial cells, cellular casts, red blood
cells and white blood cells.
HOW TO CULTURE URINE
Culture on Cysteine Lactose Electrolyte Deficiency Agar (C.L.E.D) and MacConkey agar (both
are differential agar) which differentiate between lactose and non-lactose fermenting
organisms. C.L.E.D does not allow swarming of Proteus.
A sterile wire loop is used to culture urine.
PROCEDURE:
Dip a wire loop inside the universal tube containing the urine (open the cork of the tube with the
side of your palm and keep holding the cover while you dip the wire loop into the urine) and
then inoculate your plate. The inoculation is done by introducing urine into the plate and making
a smear, from the inoculums a primary and secondary streaking is made.
To make the primary streaking, spread from the inoculums at angle 90 and the secondary
streaking is done by spreading from the primary streaking. Then incubate overnight -that is
putting inoculated plate in the incubator at 350C for 18-24 hours. The plate can then be read
the next day. On CLED, the lactose and non-lactose fermenting organisms are checked and
then confirmed on MacConkey agar.
For lactose fermenting organisms, the colonial appearance is recorded and then the gram staining
is done. If it is gram negative, the organism present could either be Klebsiellaor Escherichia coli.
Biochemical test can then be done by inoculating citrate, urea and peptone water. The peptone
water is used for sensitivity test on nutrient agar (DST); the plate is then incubated at 370C and
also the urea and citrate for 12-18 hours (overnight). Klebsiella is evident if citrate is positive or
urea is positive.
Citrate is positive when it is blue in color whereas urea is negative when it is yellow and
positive when it is red. Citrate is negative and urea is negative when Escherichia coliis evident.
FOR NON-LACTOSE FERMENTERS, oxidase test is done. Positive oxidase test shows
evidence of Pseudomonas species in urine while negative oxidase test shows evidence of
Salmonella,Shigella, Proteus,Vibrio cholerae. Biochemical test is then done for these
organisms.

3.2.2. MALARIA PARASITE TEST

Some parasites that could be detected in the blood are: Plasmodia, Trypanosomes,
Leishmania,filarial worms.
SPECIMEN-A one meter in blood diameter of the blood film of the patient. Specific
identification of parasites requires a permanent stain. For permanent staining, two types of blood
films can be prepared. Thick films allow a larger volume of blood to be examined, thus making it
easier to detect light infections with fewer parasites, while species identification is difficult. Thin
films are necessary to see the morphological characteristics of the parasites and to identify them.
PROCEDURES:
Step1
An absolutely clean,grease-free slide, well-washed slide cleaned with 70% ethanol is
recommended (at Standard Medical Diagnostics Laboratory new slides are used). The slide
is labeled with the patient’s laboratory number. Step 2
Swab the top of the patient’s third finger or thumb with cotton wool soaked with ethylated spirit
to disinfect and clean the possible micro organisms present on the surface of the skin.
Step3
Prick the point cleaned with a sterilized lancet and discard immediately.
Step 4
Apply pressure on the lower side of the top with your own hand so the blood would be able to
come out in few trickles as a drop or two will be placed on either sides of the slide since the
laboratory number labeled on the slide will be in the middle.
Step 5
You prepare a thick blood film for the malaria parasite test. To make a thick blood film, place
two or three small drops of fresh blood without anticoagulant on a clean slide with a sterilized
end of another slide. Mix the drops in acircular motion over an area about two centimeters in
diameter, (continue mixing for about thirty seconds to prevent formation of fibrin strands that
may obscure the parasites after staining, if anti- coagulated blood is used, it is not necessary to
continue mixing for thirty seconds).
Step 6
Allow the film to dry in air at room temperature on a dryer (hotplate) to fix the film.
Step 7
After drying, the slide is placed directly into an aqueous stain called Giemsa stain to make the
thick blood film to lyses the red blood cells and to remove hemoglobin so that the parasites can
be easily detected.
PRECAUTION: It is necessary for one to be very careful while collecting and preparing
blood samples. A number of parasitological, bacterial and viral diseases can be transmitted
through blood. Blood film should be prepared preferably within one hour of collection.
The time of collection should be mentioned on the specimen as well as on the result sheet and
also the laboratory number for correlation.
It is preferable to prepare blood films with fresh blood without anticoagulant. If it is not possible,
blood pant coagulated with EDTA(10mg/5ml) should be used.

GIEMSA STAINING TECHNIQUE

Giemsa stain is a Romanaosky that requires dilution in buffered water or buffered saline before
use.
Giemsa stain (stock solution)
Giemsa stain powder 0.6g
Methanol, absolute (acetone-free) 50ml
Glycerol 50ml
Dissolve the Giemsa stain in methanol in a brown bottle containing a few glass beads. Add
glycerol, mix and place the bottle in a water bath at 50-60 degree centigrade for two hours to
dissolve the stain. Shake gently at half-hour intervals. The stain should stand at room
temperature for three weeks and should be filtered before use. If kept air-tight, the stain is stable
for several months.
Preparing a working solution of Giemsa stain
For thick films the commercial stock solution is diluted with the ratio 1:50 with a neutral or
slightly alkaline buffer(7.0 to 7.2) e.g. phosphate or tap water if the pH is satisfactory.
TECHNIQUE USED
The labeled slide with the blood film on either end of it is directly stained in diluted Giemsa stain
for like 15 minutes.
It is then brought out and rinsed properly with tap water, gently flushing the stain off the slide
with water.
Dip the slide briefly in the buffer or rinse under gently running tap water.
• Wipe the under surface of the slide to remove excess stain.
• Allow it to air-dry in a vertical position.
• View under the light microscope with the oil immersion lens.
• A drop of oil is placed on each dried, stained and fixed blood film and then viewed for
malarial parasites.
TROPHOZOITE OF MALARIAL PARASITE AS VIEWED UNDER THE LIGHT
MICROSCOPE.
Trophozoite is the growing form of the parasite in the peripheral blood of man after the invasion
of the red blood cells by merozites. When the mature schizonts rupture the merozites penetrate
the red blood cells and develop into trophozoites.
Immature trophozoites are concave disc appearing as ring forms in stained preparation. It
consists of:
1. A rod-shaped nucleus (chromatin dot) stained red.
2. A peripheral rim of cytoplasm that stains blue and
3. An unstained clear area or vacuole in the centre that pushes the chromatin dot to the
periphery of the cytoplasm.
4. Three stages of the asexual life cycle occur in man, namely the trophozoites, schizont
and the gametocytes.
The parasites reside in the peripheral red blood cells. Each species is identified on two basic
parameters.
1. Appearance of the infected red blood cells.
2. Appearance of the parasite.
Results: Malaria Parasite
Chromatin of parasite: Dark red
Cytoplasm of parasite: Blue
Schuffner’s clots: Red
Maurer’s dots: Red-mauve

3.2.3. BLOOD MICROFILARIA TEST

• In this test, blood is gotten from the patient’s vein.

• A rubber tube rope is tied on the upper part of the patient’s arm.
• A vein is located between the middle fold of the arm and the upper section of the arm.
When the vein is located, the spot where it is found is swabbed with a cotton wool
soaked in spirit.
• A sterilized needle is used to prick the vein and blood is drawn and immediately
 transferred into the small anticoagulant bottle gotten from the reception, it is corked
tightly.
• Immediately you are through drawing the blood you loosen the rubber rope on the arm
to reduce pressure and the blood stops coming out.
• You clean the spot on the person’s arm after all these with a clean sterilized cotton wool
* Note the blood gotten can be used for either microfilaria test or malaria parasite test.
TEST
Blood
Microfilaria can be detected in the direct wet mount of fresh blood by their characteristics, shape
and motility.
For identification of microfilaria worm in stained blood films, the following characteristics are
looked for:
(i) Presence or absence of a sheath.
(ii) Presence or absence of nuclei at the tip of the tail
(iii) Size of microfilaria worm
(iv) Size of cephalic space in sheathed microfilaria worm.

3.2.4. STOOL MICROSCOPY

Three protozoan parasites which may be found in human stool are:
- Rhizopodea (amoebae) e.g. Entamoebahistolytica
- Zoomastigophora (flagellates) e.g. GiareliaIntesinalis
- Ciliatea(ciliates) e.g. Balantidiumcoli
EXAMININATION OF FAECES
It is viewed under light microscope at x10 and x40
First you view macroscopically for the following: Form, color, Smell, consistency, presence of
blood, and mucus, nematode, tapeworm, and segments. When viewed under the microscope in
normal saline
• You place a drop of normal saline on a thin slide with the pipette in the normal saline
bottle.
 • Pick a tiny bit of the stool sample and make a smear in the normal saline with it.
• View under the light microscope for cellular exudates such as helminthes egg, protozoa
cyst, and actual larva of nematode worms.
• When viewed under the microscope in iodine it is the same process as listed in the first
two steps above, just use iodine in this case and not normal saline.
When viewed under the light microscope, stained protozoa cysts are more visible.Other things
that could be seen under the microscope are: fat globules, undigested starch, vegetable cells,
and air bubbles. Cysts can be concentrated by the formal ether technique or by a simple
floatation in concentrated zinc sulphate

3.2.5. SEMEN ANALYSIS

Semen Analysis with Microscopy
This involves the analysis of semen by culturing and performing sensitivity test.
Part A
(i) Physical examination
• Volume: 1ml, 2ml and above
• Viscosity: Watery or Normal
• Appearance: creamy, whitish or Creamy – whitish (ii) Microscopy
• Motility
• High power
• Normal
• Abnormal
N.B: The best sperm count is about 90x106 total counts but normal count is 45 x 106, but when
the total count is 25 x 106 the diagnosis could be infertility.
(i) After the examination in part A, sterilize inoculating loop by flaming, culture the semen
sample on MacConkey and Chocolate agar.
NOTE* to always culture on chocolate agar you cut the agar in the middle and throwing of
this cut part into the waste to prevent organisms from swarming in the agar.
(ii) Incubate for 24 hours
(iii) Examine the colony if there is growth, gram stain, set up biochemical tests
(iv) Inoculate peptone water for flooding of DST (sensitivity test using the right Antibiotic
 disc).
HOW TO CARRY OUT SENSITIVITY TEST:
Flood the nutrient agar with inoculated peptone water, place the antibiotic disc on the
flooded plate and incubate overnight for 12-18 hours. At the end of the stipulated time any
antibiotic surrounded by a region where no microorganism grew can proof useful against the
microorganism discovered present. 4.7 Procedures for gram stain
(i) Crystal violet solution
(ii) Iodine solution (functions as a mordant)
(iii) Acetone (decolorizes)
(iv) Safranin (counter stain) Procedure
1. Prepare a heat fixed smear from a 18-24 hour old culture
2. Stain with crystal violet solution for 1 – 2 minutes and rinse off the solution.
3. Rinse off with iodine solution for 1 minute
4. Rinse off the iodine solution and wash the slide with acetone until the crystal violet
dye no longer runs from the slide and this will last only 5-15 seconds.
5. Rinse under gentle – running tap, and counter stain with safranin for 30 seconds.
6. Wash with water, blot dry and examine under microscope.
Observation
1 Gram-positive cell appear purple, or crystal violet iodine complex
2 Gram negative cells are red or pink
Note: Cells could be either bacilli or cocci.
 CHAPTER FOUR

4.1. HAEMATOLOGY LABORATORY

In this laboratory, the following tests are carried out
• Blood Group
• Pregnancy Test
• Widal Agglutination Reaction
• Packed Cell Volume
• Human Immune Deficiency Virus (HIV) Screening

4.1.1. BLOOD GROUP TEST

Analysis
A needle is inserted into the vein and blood into a tube. During the procedure, the elastic band
used is reserved to restore circulation. Once the blood has been collected, the needle is
removed and a band aid or gauze is applied.

Procedures

• Venous blood is collected into EDTA sample bottle

• Antiserum A, B, and D were placed on the white tile separately in three spots
• Three separate drops of blood were dropped unto each of the spots
• Each spot was then mixed together with the tip of a clean glass slide or an inverted rubber
pipette
• The tile was rocked for three minutes to view agglutination
Results

Blood type (or blood group) is determined, in part, by the ABO blood group antigens present
on red blood cells.
A blood type (also called a blood group) is a classification of blood based on the presence or
absence of inherited antigenic substances on the surface of red blood cells (RBCs). These
antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the
blood group system. Some of these antigens are also present on the surface of
Red blood cell compatibility
• Blood group AB individuals have both A and B antigens on the surface of their RBCs,
and their blood serum does not contain any antibodies against either A or B antigen. Therefore,
an individual with type AB blood can receive blood from any group (with AB being preferable),
but can donate blood only to another type AB individual.
• Blood group A individuals have the A antigen on the surface of their RBCs, and blood
serum containing IgM antibodies against the B antigen. Therefore, a group A individual can
receive blood only from individuals of groups A or O (with A being preferable), and can donate
blood to individuals with type A or AB.
• Blood group B individuals have the B antigen on the surface of their RBCs, and blood
serum containing IgM antibodies against the A antigen. Therefore, a group B individual can
receive blood only from individuals of groups B or O (with B being preferable), and can donate
blood to individuals with type B or AB.
•Blood group O (or blood group zero in some countries) individuals do not have either A or B
antigens on the surface of their RBCs, but their blood serum contains IgM anti-A antibodies and
anti-B antibodies against the A and B blood group antigens. Therefore, a group O individual can
receive blood only from a group O individual, but can donate blood to individuals of any ABO
blood group (i.e. A, B, O or AB). If anyone needs a blood transfusion in a dire emergency, and
if the time taken to process the recipient's blood would cause a detrimental delay, O Negative
blood can be issued.
RBC Compatibility chart
In addition to donating to the same blood group; type O blood donors can give to A, B and AB;
blood donors of types A and B can give to AB.
Red blood cell compatibility table

Recipient[1] Donor[1]

O− O+ A− A+ B− B+ AB− AB+

O−

O+

A−

A+

B−

B+

AB−

AB+

Table note
1. Assumes absence of atypical antibodies that would cause an incompatibility between donor
and recipient blood, as is usual for blood selected by cross matching.
Recipients can receive plasma of the same blood group, but otherwise the donor-recipient
compatibility for blood plasma is the converse of that of RBCs: plasma extracted from type AB
blood can be transfused to individuals of any blood group; individuals of blood group O can
receive plasma from any blood group; and type O plasma can be used only by type O
recipients.
Plasma compatibility table

Recipient Donor[1]

O A B AB

AB

Table note
1. Assumes absence of strong typical antibodies in donor plasma

Rhesus D antibodies are uncommon, so generally neither D negative nor D positive blood
contain anti-D antibodies. If a potential donor is found to have anti-D antibodies or any strong
atypical blood group antibody by antibody screening in the blood bank, they would not be
accepted as a donor (or in some blood banks the blood would be drawn but the product would
need to be appropriately labeled); therefore, donor blood plasma issued by a blood bank can be
selected to be free of D antibodies and free of other atypical antibodies, and such donor plasma
issued from a blood bank would be suitable for a recipient who may be D positive or D
negative, as long as blood plasma and the recipient are ABO compatible.

4.1.2. PREGNANCY TEST

PRECAUTION
It is necessary for one to be very careful while collecting and preparing blood samples. A
number of parasitological, bacterial and viral diseases can be transmitted through blood..
The time of collection should be mentioned on the specimen as well as on the result sheet and
also the laboratory number for correlation.
STEPS INVOLVED
1. Select a sterile, dry plastic syringe of the capacity required, e.g. 2.5ml,5ml or 10ml
2. Apply a soft tubing tourniquet or Velcro arm bound to the upper arm of the patient
3. Using the index finger feel for a suitable vein, selecting a sufficiently large straight vein that
does not roll and with a direction that can be felt.
4. Cleanse the puncture site with 70% ethanol and allow drying.
5. When sufficient blood has been collected, release the tourniquet and instruct the patient to
open his or her fist.
6. Centrifuge for 3-5 minutes (RCF 12000-15000xg), using the shorter time when the RCF is
15,000xg
7. Immediately after centrifuging, first check that there has been no leakage of blood from the
bottle or breakage.
8. Pregnancy Test is therefore carried out by inserting a pregnancy strip in the bottle containing
blood.
Most chemical tests for pregnancy look for the presence of the beta subunit of hCG or
human chorionic gonadotropin in the blood or urine . hCG can be detected in urine or blood
after implantation, which occurs six to twelve days after fertilization.[1] Quantitative blood
(serum beta) tests can detect hCG levels as low as 1 mIU/mL, while urine tests have published
detection thresholds of 20 mIU/mL to 100 mIU/mL, depending on the brand.[7] Qualitative
blood tests generally have a threshold of 25 mIU/mL, and so are less sensitive than some
available home pregnancy tests. Most home pregnancy tests are based on lateral-flow
technology.
RESULTS
The strip shows whether the patient is pregnant or not if
• Positive (double line): the patient is pregnant
• Negative (single line): the patient is not pregnant
• Invalid: No visible band at all. The test is repeated
NOTE: For detection of hCG in urine, same procedure is followed
Precautions
• Test kit must not be beyond expiry date
• The test device must not be reused
 • The test kit is for in vitro diagonostic use only

4.1.3. WIDAL AGGLUTINATION REACTION

Materials

Widal kit, white tile, pipette, test tube, centrifuge, stop watch, blood sample

Procedure

• Venous blood is collected into sample bottle and spun at 3000rpm for 5 minutes
• The serum is taken with the aid of a pipette and put on white tile in different spots of 4
per row making two rows
• First row is labeled O, OA, OB, OC and the second row H, HA, HB, HC respectively.
• Antiserum from the widal kit for each spot are released on top of the pipette blood.
• The tile is then rocked for 3 minutes

Results

Expected results ratio:

• Highly reactive……….1:320(positive)
• Very reactive…………1:160(positive)
• Weak reaction………..1:80(positive)
• Non Significant………1:40(negative)
• Non Significant………1:20(negative)

4.1.4. PACKED CELL VOLUME

Value of Test:
The packed cell volume also called haematocrit, is used to calculate the mean cell
haemaglobinconcentration (MCHC) and mean cell volume (MCV). These red cell indices are
used in the investigation of anaemia. The PCV is also used to screen for anaemia when it is not
possible to measure hemoglobin, and to diagnose polychaemiavera and to monitor its
treatment.

It is suitable for screening large clinic populations’e.g. antenatal clinics.

Specimen:
To measure the PCV, either well mixed well oxygenated EDTA anticoagulated blood can be
used or capillary blood collected into a heparinzed capillary.
Equipment:
Microhaematocrit reader, centrifuge, needle, syringe, capillary tube.
Test Method
1.About three quarters fills either
• a plain capillary with well mixed EDTA anticoagulated blood(tested within 4 hours of
collection), or
• a heparinised capillary with capillary tube
2. Seal the unfilled end, preferably using a sealant material.If unavailable, heats seal the
capillary using a small flame from a sprint or a pilot flame of a bursen burner, rotating the end
of a capillary in the flame.
3. Carefully locate the capillary in one of the numbered slots of the microhaematocrit rotor
with the sealed end against the rim gasket (to prevent breakage). Write the number on the
patient form.
4. Centrifuge for 3-5 minutes (RCF 12000-15000xg), using the shorter time when the RCF
is 15,000xg
5. Immediately after centrifuging, read the PCV. First check that there has been no leakage
of blood from the capillary or breakage. To read the PCV in a hand held, align the base of the
red cell column on the 0 line and the top of the plasma column on the 100line.Read off the PCV
from scale. The reading point is the top of the red cell column, just below the buffy coat layer
(consisting of WBCs and platelets).
Results
Above the packed red cells is a white layer of platelets. Plasma is usually straw colored, but if
bright yellow;it is jaundiced, when colorless; it is iron deficient, when red; haemolysis has
occurred
The normal PCV range for male is 39% - 53%.
The normal PCV range for female is 35% -49%.
Factors that affect PCV result
• Quality of capillary tube
• Time and speed of centrifugation
• Spectrum collection: quantity of anticoagualant

4.1.5. HUMAN IMMUNE DEFICIENCY SCREENING

HIV tests are used to detect the presence of the human immunodeficiency virus in serum, saliva,
or urine. Such tests may detect HIV antibodies, antigens, or RNA.
Terminology
The window period is the time from infection until a test can detect any change. The average
window period with HIV-1 antibody tests is 22 days for subtype B. Antigen testing cuts the
window period to approximately 16 days and NAT (Nucleic Acid Testing) further reduces
this period to 12 days.
Performance of medical tests is often described in terms of:
• Sensitivity: The percentage of the results that will be positive when HIV is present
• Specificity: The percentage of the results that will be negative when HIV is not present.
All diagnostic tests have limitations, and sometimes their use may produce erroneous or
questionable results.
• False positive: The test incorrectly indicates that HIV is present in a non-infected person.
• False negative: The test incorrectly indicates that HIV is absent in an infected person.
Nonspecific reactions, hypergammaglobulinemia, or the presence of antibodies directed
to other infectious agents that may be antigenically similar to HIV can produce false
positive results. Autoimmune diseases, such as systemic lupus erythematosus, have also
rarely caused false positive results. Most false negative results are due to the window
period; other factors, such as post-exposure prophylaxis, can rarely produce false
negatives.
Rapid Antibody Tests are qualitative immunoassays intended for use as a point-of-care test to aid
in the diagnosis of HIV infection. These tests should be used in conjunction with the clinical
status, history, and risk factors of the person being tested. The specificity of Rapid Antibody
Tests in low-risk populations has not been evaluated. These tests should be used in appropriate
multi-test algorithms designed for statistical validation of rapid HIV test results.
If no antibodies to HIV are detected, this does not mean the person has not been infected with
HIV. It may take several months after HIV infection for the antibody response to reach
detectable levels, during which time rapid testing for antibodies to HIV will not be indicative
of true infection status. For most people, HIV antibodies reach a detectable level after two to
six weeks. Materials
Blood serum, Abort determine HIV-1 and HIV-2 test kit, and centrifuge
Procedure
• Venous blood is collected into EDTA sample bottle
• The blood is spun at 3000rpm for 10 minutes
• The strip is then immersed into blood serum with the narrow end pointing towards the
blood
• It must be immersed past the mark line. The strip is taken out after 3 seconds and laid on
a flat clean dry non absorbent surface.
• Water for colored band to appear Results
Readings should be taken within 10 minutes
• Positive: Distinct color band appear on the control and test regions. This indicates the
presence of HIV-1 and HIV-2
• Negative: Only one color band appears on the control region. No apparent band on the
test region. This indicates that the patient is HIV negative
• Invalid: No visible band at all. The test is repeated
 CHAPTER FIVE

5.1 CHEMICAL PATHOLOGY

The following tests are carried out in Chemical pathology laboratory
• Fasting Blood Sugar
• Random Blood Sugar
5.1.1. FASTING BLOOD SUGAR (F.B.S).
The fluctuation of blood sugar (red) and the sugar-lowering hormone insulin (blue) in
humans during the course of a day with three meals. One of the effects of a sugar-rich vs a
starch-rich meal is highlighted.
The blood sugar concentration or blood glucose level is the amount of glucose (sugar) present in
the blood of a human or animal. Normally, in mammals the body maintains the blood glucose
level at a reference range between about 3.6 and 5.8 mM (mmol/L). It is tightly regulated as a
part of metabolic homeostasis.
Materials:
Blood sample, glucomter
NOTE: Glucometer is an instrument used to measure the glucose(sugar) level of a patient.
Procedure
• Blood is collected from the thumb of the patient
• The blood is made to drop at the tip end of the glucometer and then left for few
minutes(about 3-5minutes)
• The reading is then taken and written down
Results
The normal range is 70-100mg/dL. If the result from the reading is very much less than
70mg/dL, the patient is said to be hypoglycemic and needs sugar transmission, if the result is far
higher than 100mg/dL the patient is said to be hyperglycemic and needs insulin transfusion.

5.1.2. RANDOM BLOOD SUGAR (R.B.S).

This test is similar to fasting blood sugar, the difference being that the test can be carried out
anytime on a patient (that is, whether the patient has or has not eaten is irrelevant) and it is
useful in the case of emergency.
Materials
Blood sample,glucometer
Procedure
• Blood is collected from the thumb of the patient
• The blood is made to drop at the tip end of the glucometer and then left for few
minutes(about 3-5minutes)
• The reading is then taken and written down.
Results
The normal range is 100-180mg/dL. If the result from the reading is very much less than
70mg/dL, the patient is said to be hypoglycemic and needs sugar transmission, if the result is far
higher than 100mg/dL the patient is said to be hyperglycemic and needs insulin transfusion
 CHAPTER SIX

6.1. EXPERIENCE GAINED

 I learnt almost all the practical aspects involved in medical Biochemistry and also
microbiology in general.

 I got to know about and learnt the use of the laboratory equipment.

 I learnt to obey all laboratory rules for my safety and that of the patients.

 I learnt to relate properly with other co-workers.

6.2. PROBLEMS ENCOUNTERED

 In most cases, safety rules are not taken into consideration and the necessary safety
gadgets and equipment are not usually in place.

 It is suggested that some form of allowance should be given to the students by the
employers as a form of encouragement and to assist in their cost of living, basically
feeding, transportation and accommodation especially in areas far from the students’
neighborhood.

 I also would want to say that more time should be given to students for their SIWES
program.

6.3. RECOMMENDATIONS
• I propose that more time should be given to the students of Biochemistry for SIWES
activities
• I recommend that government should provide placements for students undergoing
SIWES in the several fields of Nigerian Economy.
• I recommend that more preference should be given to the power sector so as to provide
adequate light to various Medical laboratories in the country.

6.4. CONCLUSION
In conclusion this program has enabled students to gain a lot and many can now practice the
applied aspects of their various disciplines and other related areas on their own. The program
has really been.