First Professional Questions and Answers by Hon. Ukpor Iyang

HON.
UKPOR IYANG IYANG (NIMELSSA PIKIN)
FIRST PROFESSIONAL QUESTIONS AND ANSWERS

First professional questions and answers is designed as an all-in-one review and
summary of the major clinical laboratory science content areas generally taught in
the departmental laboratory practical. It is developed to help examination
candidates prepare for laboratory posting (practical) examination and First
professional examination.
This Document is dedicated to Medical laboratory science students everywhere.
In your upcoming role as professionals, remember that your job is important to
the medical world as well as to the individual patient. Be proud of the fact that
you will be making a difference in people’s lives. Always be excited about the
unlimited opportunities available in your chosen profession and help lead the
field of Medical Laboratory Science well into the 21st century.
However, it should be noted that reading this document is at readers risk as it is
compiled by a student in his own perspective point of comprehension, therefore
the reader is prone to come across mistakes. It is therefore advised to verify the
text and other reference values.
Any suggestions and constructive criticisms from the students alike to enrich the
guide in future are very much welcomed.
PARASITOLOGY
QUESTION 1
Sample A is a blood specimen collected from a patient who presented with a clinical
manifestation of fever, headache, cold etc. prepare a thick film of the specimen for the
detection of malaria parasite.
ANSWER (1)
DATE: 24/10/2017
TITLE: IDENTIFICATION OF MALARIA PARASITE
1
CAPACITY THROUGH SERVICE, THE WAY FORWARD FOR NIMELSSITES
HON. UKPOR IYANG IYANG (NIMELSSA PIKIN)
AIM: To identify the malaria parasite in sample marked “A”

METHOD: Giemsa staining technique
PRINCIPLE: This is based on the principle of Romanowsky stains; Giemsa stain contains
methylene blue (basic dye) which stains the acidic components of the cell (nuclear DNA,
cytoplasmic RNA) bluish-purple and Eosin (acidic dye) which stains the basic component of the
cell (hemoglobin and granules in eosinophil) orange to pink.
MATERIALS: Microscope slide, Pasteur pipette, cotton wool, light microscope, hot air oven,
immersion oil.
PROCEDURE:
1. A Pasteur pipette was used to pipette a certain quantity of the well mixed sample
labelled A. then a drop of the sample was placed on a well labelled clean grease-free
slide.
2. A thick firm was made by smearing the blood on the slide with head of the pipette and
the slide was allowed to air dry.
3. Then the slide was flooded with a 1 in 30 dilution of Giemsa stain for 30min.
4. After staining, the slide was rinsed in water and blotted dry with cotton wool. Then the
slide was allowed to air dry.
5. After drying, a drop of immersion oil was placed on the stained portion of the slide and
it was viewed under the light microscope using 40x and 100x objective lens to focus and
view respectively with the iris diaphragm opened and condenser rack up.
NOTE:
i. If a hot air oven was provided for drying, you can replace “air drying” with “drying in
the oven”
ii. If field stain is provided instead of Giemsa, indicate in the report.
iii. Should incase Giemsa stain is provided, find out the dilution factor for the stain and
reflect it in the report
• 1 in 10 dilution factor = 10min staining
• 1 in 20 dilution factor = 20 min staining
• 1 in 30 dilution factor = 30 min staining
RESULT:
Chromatin of parasite . . . . . . . . . . . . . . . . Dark red Cytoplasm
of parasite . . . . . . . . . . . . . . . . . . . . Blue
CONCLUSION:
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PRECAUTIONS:
1. I ensured accurate and proper timing during staining 2.

I ensured that the smear made was not too thick.
QUESTION 2
Examine the stool sample marked G using saline-iodine method for the identification of
parasites.
ANSWER (2)
DATE: 27/11/2017
TITLE: STOOL ANALYSIS
AIM: To identify intestinal parasites present in the sample marked G.
METHOD: Saline-iodine method
MATERIALS: Normal saline, iodine, clean glass slide, applicator stick, cover slip, Pasteur pipette
and microscope.
A. MCROSCOPY
PROCEDURE
1. The stool sample was physically examined for its appearance, colour and consistency
2. The presence of pus, mucus, worms and blood was also observed for.
RESULT:
COLOUR yellowish-brown
CONSISTENCY formed
PUS (yellowish-whitish) absent
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WORMS absent
BLOOD absent
B. MICROSCOPY
PROCEDURE
1. A drop of normal saline was placed on one end of a slide while a drop of iodine was
placed on the other end of the slide.
2. The stool sample was picked with the aid of an applicator stick and dropped on both
ends of the slide containing the normal saline, then iodine.
3. The stool sample was emulsified on both ends and covered with cover slip.
4. It was mounted on a light microscope. The wet preparation were focused on with
10x objective lens and viewed with 40x objective lens.
RESULT:
 No ova, cyst or vegetative form of parasite seen

 E.g Ova of Ascaris lumbricoides seen (if observed)
CONCLUSION
QUESTION 3
Sample F is a stool specimen collected from a 2 year old child residing in AKTH. Examine the
sample using Formol ether concentration technique for the presence of ova, larvae and cysts,
ANSWER (3)
DATE: 20/DEC/2017
TITLE: STOOL ANALYSIS
4
AIM: To identify intestinal parasites present in the sample labelled F.

METHOD: Formol ether concentration technique
PRINCIPLE: In the Ridley modified method, faeces are emulsified in formol water, the
suspension is strained to remove large faecal particles, ether or ethyl acetate is added, and the
mixed suspension is centrifuged. Cysts, oocysts, eggs, and larvae are fixed and sedimented and
the faecal debris is separated in a layer between the ether and the formol water. Faecal fat is
dissolved in the ether.
MATERIALS: Applicator stick, screw-cap tube, 10% v/v formol water, beaker, centrifuge
(conical) tube, diethyl ether (or ethyl acetate), tissue, centrifuge, microscope, glass slide, cover
slip.
PROCEDURE:
1. An applicator stick was used to emulsify an estimated (1g/pea size) of faeces in about
4ml of 10% formol water contained in a screw-cap tube.
Note: Include in the sample, faeces from the surface and several places in the specimen.
2. A further 4ml of 10% v/v formol water was added and mixed well by shaking.
3. The emulsified faeces was sieved and the sieved suspension was collected in a beaker.
4. The suspension was then transferred to a centrifuge (conical) tube.
5. 4ml of diethyl ether (or ethyl acetate) was added
6. The tube was stoppered and mixed for 1minute
7. A tissue (or piece of cloth wrapped around the top of the tube) was used to loosen the
stopper (considerable pressure will have built up inside the tube).
8. The suspension was Centrifuged immediately at 3000 rpm) for 1 minute.
9. After centrifuging, the parasites will have sedimented to the bottom of the tube and the
faecal debris will have collected in a layer between the ether and formol water
10. Using a stick (or the stem of a plastic bulb pipette), the layer of faecal debris was loosen
from the side of the tube and the tube was inverted to discard the ether, fecal debris
and formol water. The sediment remained.
11. The tube was returned to its upright position and the fluid was allowed to drain from
the side of the tube to the bottom
12. The bottom of the tube was tapped in order to resuspend and the sediment was mixed
well.
13. The sediment was transferred to a slide and cover slipped.
14. The preparation was examined microscopically using 10X objectives with the condenser
iris closed sufficiently to give good contrast and 40X objective to examine small cysts
and eggs.
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NOTE:
 To assist in the identification of cysts, run a small drop of iodine under the cover glass
 Although the motility of Strongyloides larvae will not be seen, the non-motile larvae can
be easily recognized.
 If required, count the number of each species of egg in the entire preparation. This will
give the approximate number per gram of faeces.
RESULT: e.g egg of Ascaris lumbricoides seen, trophozoite of E.histolytica seen, Adult worm of
E. vermicularis seen.
CONCLUSION:
Parasites found in faeces

CLASIFICATION PARASITE FORM
Amoeba E. histolytica cyst, trophozoite
Intestinal flagellates G. lamblia cyst, trophozoite
Ciliates B. coli cyst, trophozoite
Sporozoans I. belli oocyst
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C. parvum oocyst
Microsporidia spores
Intestinal nematodes S. stercoralis Larva
E. vermicularis worm
N. americanus egg
A. duodenale egg
T. trichiura egg
A. lumbricoides egg, worm
trematodes F. hepatica egg
F. gigantica egg
F. buski egg
Schistosoma spp egg
Paragonimus spp egg
H. heterophyes egg
M. yokagawai egg
cestodes T. saginata egg, segment
T. solium egg, segment
V. nana egg
D. latum egg
Parasites found in urine and blood

urine schistosoma haematobium Egg
urine Trichomonas vaginalis Trophozoite
Blood Plasmodium species
Blood Leishmania species
Blood Trypanosomes
QUESTION 4
Prepare two films from N, stain one by leishmans stain and comment fully on the blood
picture. (Differential count not required) and leave the other unstained on the bench.
ANSWER (4)
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DATE: 24/10/2017
TITLE: THIN FILM MAKING

AIM: To prepare thin film and determine the cell morphology of the sample labelled N
METHOD: lieshman staining technique
PRINCIPLE: this is based on the principle of Romanowsky stains. Leishmann stain contains
methylene blue (basic dye) which stains acidic components of the cell bluish purple and Eosin
(acidic dye) which stains the basic component of the cell orange to pink.
MATERIALS: Lieshman stain, glass slide, whole blood, Pasteur pipette, light microscope, water.
PROCEDURE:
FILM MAKING
1. Sample N was mixed properly

2. Using a Pasteur pipette, a drop of sample A was placed 1cm away from the edge of a
clean grease free glass slide
3. A spreader was placed at the edge of the blood, the blood was allowed to extend along
the edge of the spreader and was drawn backward to touch the blood
4. At an angle of 45o, the blood was then pushed forward to make a thin film with a
smooth head, body and tail.
5. The blood firm was allowed to air dry properly, there after immersion oil was placed on
the stained portion of the slide
6. The film was examined under the light microscope using 40X objective and 100X to
focus and to view.
7. One of the slides was left on the bench for inspection
STAINING
1. Lieshman stain was flooded on the other thin film placed on the staining rack and was
allowed to stained for 2min
2. It was diluted twice volume of the stain using buffered distilled water of pH 6.8 and was
left for 8min
3. It was then washed off using the buffered distilled water of pH 6.8
4. It was drained and air dried
5. It was examined under the microscope using oil immersion objective (100X)
RESULT:
 Red cells………….pink-red
 Nucleus of cells………..purple-violet
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Cytoplasm
 Neutrophils, eosinophils………….pale pink

 Large lymphocytes……..clear blue
 Small lymphocytes……….darker clear blue
 Monocytes…….grey blue
Granules
 Eosinophils………orange red
 Neutrophils……..mauve blue
 Toxic granules……..dark violet
 Basophils………..dark blue – violet
 Platelets------purple blue
Inclusions
 Malaria pigment (in monocytes)………brown – black

 Howell-jolly body………purple-violet
Cells
 Macrocyte i.e when cell diameter > 8µm

 Microcyte –i.e cell diameter < 6 µm
 Hypochromia – increased zone of central pallor
 Polychromatophilia – presence of red cells not fully haemoglobinised
 pokilocyte – variability of cell size
 Schistocyte – presence of cell fragments in circulation
 Elliptocyte – oval cells
 Spherocyte - cell without central parlour, loss of biconcave shape etc
CONCLUSION:
CLINICAL SIGNIFICANCE:
Examination of thin blood film is important in the investigation and management of anemia,
infections and other conditions which produce changes in the appearance of blood cells and
different white cell count
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QUESTION 5
Sample A is a blood specimen collected from a patient who presented with a clinical
manifestation of fever, headache, cold etc. prepare a thick film and thin film of the specimen
for the detection of malaria parasite.
ANSWER 4
DATE: 24/10/2017
TITLE: IDENTIFICATION OF MALARIA PARASITE
AIM: To identify the malaria parasite in sample labelled “M”
MATERIALS: Glass slide, staining rack, field stain A and B, pipette, methanol, draining rack,
distilled water, cotton wool,
METHOD 1: THIN FILM STAINING TECHNIQUE
PROCEDURE:
1. A thin film was made

2. The slide was placed on a staining rack and the methanol-fixed thin film was flooded
with 0.5ml of diluted field stain B
3. Add Equal volume of field stain A and mix with the diluted field stain B 4. It was
allowed to stain for 1minute.
Note: The stains can be easily applied and mixed on the slide by using 1ml graduated plastic
bulb pipettes.
5. The stain was washed off with distilled water. The back of the slide was wiped clean and
placed on the staining rack and the film was allowed to air dry.
6. It was examined under the microscope using oil immersion objective (100X)
RESULT:
Chromatin of parasite . . . . . . . . . . . . . . . . Dark red
Cytoplasm of parasite . . . . . . . . . . . . . . . . . . . . Blue
Malaria pigment in white cells. . . . . . . Brown-black Red
cells. . . . . . . . . . . . . . Grey to pale mauve-pink
Reticulocytes . . . . . . . . . . . . . . . . . . . . . . . Grey-blue
Nuclei of neutrophils . . . . . . . . . . . . . . . Dark purple
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Granules of eosinophils . . . . . . . . . . . . . . . . . . . Red

METHOD 2: THICK FILM FIELD’S STAINING TECHNIQUE
PROCEDURE:
1. A thick film was made on a slide.

2. The slide was dipped into field stain A for 5 secs. The excess stain was drained off by
touching a corner of the slide against the side of the container.
3. The slide was washed off gently for about 5secs in a clean water. The excess water was
drained off.
4. The slide was dipped into field stain B for 3 seconds. The excess stain was drain.
5. The slide was washed gently in clean water. The back of the slide was wiped clean and
it was placed on the draining rack and it was allowed to air dry.
N0TE: If after staining, the whole of the film appears yellow- brown (usually a sign that too
much blood has been used), too blue, or too pink, do not attempt to examine it. Re-stain it by
dipping the slide into Field’s stain A for 1 second, followed by a gentle wash in clean water, dip
into Field’s stain B for 1 second and a final gentle wash in clean water.
RESULT:
Results for malaria thick film
Chromatin of parasite . . . . . . . . . . . . . . . . . Dark red
Cytoplasm of parasite ……………………………………………………. Blue-mauve
P. vivax and P. ovale parasites
Malaria pigment ………………………………………………...Yellow-brown or brown-black
STUDY QUESTIONS
1 A 2 year old male subject reported to the AKTH with a history of diarrhoea, vomiting
and abdominal tenderness.
a. With the clinical details provided, establish a clinical diagnosis and name the
appropriate type of sample to be used for the laboratory diagnosis.
b. You are provided with a specimen labelled K, identify the parasites as far as
possible with the materials provided.
HINT: The clinical diagnosis might be giardiasis, amoebiasis, colitis or any intestinal
parasitic infection. The sample to use here is stool.
2 Sample G is a stool sample. Perform a direct microscopy of the sample for intestinal
parasites. Report your findings
3 Sample K is a stool sample obtained from a subject with history of colitis. Using the
materials provided. Examine the stool sample provided and report your findings
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4 The specimen labelled C is a blood sample collected from a subject suffering from a
fever. Prepare a blood film, stain the blood film with the reagents provided and report
your findings.
BACTERIOLOGY
QUESTION 1
You are provided with a chocolate agar plate marked C. carryout the following test and report
your findings:
a. Describe the appearance of the bacterial colony.

b. Make a smear from the culture and stain by grams method. Comment on your result.
c. Carryout simple tests to determine whether or not any of the organisms identified are
motile.
ANSWER (1.a)
DATE: 01 /5/2015
TITLE: IDENTIFICATION OF BACTERIAL COLONY
AIM: to identify the bacterial organism present in plate labelled C using scocosheep
METHOD: colonial morphology
MATERIALS: Cultured plate, sterile wire loop, ruler
PROCEDURE:
1. The given cultured plate was used to observed the largest colonies with the naked eye to
determine their various characteristic
RESULT:
S/N CHARACTERISTICS COLONY
1 size 0.5 in diameter
2 colour white
3 odour fecal odour
4 consistency smooth
5 opacity translucent
6 shape circular
7 hemolysis non alpha, non-beta
8 elevation raised
12
9 edge regular
10 pigmentation absent
NOTE: These results are assumptions.
DIAGRAM:
CONCLUSION:
ANSWER (1b)
TITLE: GRAM STAINING
AIM: to differentiate between Gram positive and Gram negative bacteria.
PRINCIPLE: The cell wall of gram positive bacteria have high amount of peptidoglycan and also
contain acidic component called teichoic acid which have high affinity for basic dye and resist
decolorization. Following staining with a basic dye and treated with iodine, the dye-iodine
complex is easily removed from the more permeable cell wall of gram positive bacteria. Upon
treatment with acetone, gram positive bacteria retain the crystal violet iodine complex
appearing purple when viewed under light microscope. On the other hand, gram negative
bacteria possess a thin peptidoglycan layer and lipopolysaccharide in their outer membrane,
thus upon treatment with acetone, the cell wall shrinks as the lipids in the cell wall dissolves
and then retain the colour of the secondary stain, neutral red appearing pink when viewed
under the light microscope.
MATERIALS: As provided (e.g crystal violet, lugols iodine, 10% acid alcohol, neutral red, slide,
staining rack, drying rack, , Bunsen burner, tap water, Cultured plate, wire loop, normal saline,
Pasteur pipette, marker (always circle your smear)
PROCEDURE:
SMEARING
1. The wireloop was sterilized to red hot and allowed to cool.

2. A Pasteur pipette was used to drop a normal saline at the centre of a grease free glass
slide
3. A small portion of bacterial growth was picked using the wireloop and emulsified in the
drop of normal saline until a thin homogenous film was made. It was allowed to air dry
4. The smear was then fixed by rapidly passing it over a Bunsen flame three times.
STAINING
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1. The fixed smear placed on a staining rack and the slide was flooded with crystal violet
for 1minute and then rinsed with water.
2. The slide was flooded with iodine for 1minute and then rinsed with water.
3. The slide was flooded with acetone for few seconds and immediately rinsed with water
4. Finally the slide was counterstained with a neutral red for 2min, or safranin 30sec, or
dilute Carbol fuschsin 12min (depending on the one given). it was rinsed with water and
blotted dry
5. The slide was then air dried
it was examined under the light microscope using 40X and 100X objective lens to focus
and view respectively.
RESULT:
Report your observation based on colour, arrangement and shape (CAS) e.g
3 colour of gram reaction pink
1 Arrangement single
2 shape rod
COMMENT: it was observed that sample C contains gram negative bacteria (Bacilli).
ANSWER (1c)
 Ask for the overnight broth culture of the culture plate (purity plate)
TITLE: MOTILITY TEST

AIM: to detect the ability of bacteria to move from one place to another.
METHOD: Hanging drop
MATERIALS: grease free glass slide, cover slip, overnight broth culture, microscope, plasticine
PROCEDURE:
1. Plasticine was used to make a ring of about 2cm in diameter on a clean grease free glass
slide
2. A drop of a well-mixed overnight broth culture was placed on the center of a coverslip.
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3. The ring of plasticine was carefully pressed on the coverslip containing the broth
culture.
4. It was inverted immediately so that the coverslip was on top.
5. It was then focused and viewed under the microscope using 10X, then 40X objective
respectively with the condenser low and iris sufficiently closed.
RESULT:
Motility positive (motile organism present)
Motility negative (motile organism absent i.e if you don’t observe motility) 
Call the supervisor for initials.
CONCLUSION: True motility was seen which indicate the presence of gram negative bacilli.
QUESTION 2
Specimen C is a urine sample collected from a 21year old female student of Bayero University
Kano with a history of urinary tract infection.
a. Centrifuge the urine specimen, examine the deposit and report your findings. culture
the sample on the MacConkey agar provided: leave on the bench
b. Gram stain the urine deposit and report your findings.
ANSWER (2.a)
DATE: 24/11/2016
TITLE: URINE MICROSCOPY
AIM: To identify the urinary parameters present in the sample labelled C
METHOD: Direct wet preparation
MATERIALS: clean grease free glass slide, cover slip, microscope, centrifuge, test tubes.
PROCEDURE:
1. A quantity of the urine was aseptically transferred into a centrifuge tube.

2. The centrifuge tube was placed in a bucket centrifuge and balanced properly.
3. The urine sample was spun at 12,000 rpm for 5min
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4. Then, the supernatant was decanted by quick inversion and the sediment poured on a
clean grease free glass slide and covered with a cover glass.
5. It was then mounted on a light microscope and the wet preparation was focused with
10X objective lens and viewed with 40X objective lens.
RESULT:
 Pus cells……………….…… seen

 Epithelial cells….…………….seen
 Red blood cells..........………seen
 No ova of parasite seen, ovum of schistosoma haematobium seen
 Other parameters……….absent
TITLE: PLATING/CULTURING
AIM: To obtain discrete colonies of the organism growing in sample labelled C MATERIALS:
wireloop, sample C, burnsen burner, hot air oven, MacConkey agar plate
PROCEDURE:
1. The agar plate was dried in the oven to reduce the moisture
2. The wire loop was flamed to red hot
3. A loop was picked from the urine deposit
4. the urine was inoculated into the MacConkey agar and was spread by means of
streaking in a unique manner as follows:
 A pool was made 1cm away from the edge of the plate
 It was then flamed and streaked with straight lines
 The wire loop was flamed again and streaked with endpoint of the first one
overlapping the line.
 The wire loop was flamed again and was streaked as above to produce different
side streaking with one zig-zag line from the last line toward inside. NOTE: You
must draw the streaking
DIAGRAM:
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ANSWER (2.b)
TITLE: GRAM STAINING TECHNIQUES
AIM: to differentiate between gram positive and gram negative bacteria.
staining rack, drying rack, , Bunsen burner, tap water, sample urine).
PROCEDURE:
1. The centrifuged urine was poured on a clean grease free glass slide and a smear was
made.
2. The smear was air dried and then heat fixed by passing the slide over the flame of a
Bunsen burner.
3. The slide was flooded with crystal violet for 1minute and then rinsed with water.
4. The slide was flooded with iodine for 1minute and then rinsed with water.
5. The slide was flooded with acetone for few seconds and immediately rinsed with water
6. Finally the slide was counterstained with a neutral red for 2min. it was rinsed with water
and blotted dry
7. The slide was then air dried
it was examined under the light microscope using 40X and 100X objective lens to focus
and view respectively.
RESULT:
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Report your observation based on colour, arrangement and shape (CAS) e.g
1 Colour of gram reaction purple
2 Arrangement cluster
3 Shape cocci
CONCLUSION: it was observed that sample C contains gram positive bacteria (Staphylococcus
species).
PRECAUTION:
 I avoided over decolorization with acetone

 I ensured accurate timing throughout the staining procedures
QUESTION 3
You are provided with an overnight culture of MacConkey agar marked B
a. Carryout a gram staining technique on different, colonies present on the plate.

b. Determine whether any of the isolates express the production of pathogenic enzyme.
ANSWER (3a)
DATE: 20/12/2020
PRINCIPLE: see previous
Pasteur pipette.
PROCEDURE:
See previous procedures above
RESULT:
Report your observation based on Colour, arrangement and shape (CAS) e.g
Number of colonies =?
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S/N CHARACTERISTICS COLONY 1 COLONY 2 COLONY 3

1 colour of gram reaction purple purple pink
2 arrangement single cluster single
3 shape cocci cocci rod
COMMENT: it was observed that sample C contains predominantly gram positive bacteria and
some colonies of gram negative bacteria
ANSWER (3b)
Assuming these materials are provided (3% hydrogen peroxide, human or rabbit plasma, kovac
reagent)
Ask for a pure culture of the mixed culture plate provided, if not given. Then carry out the
following test:
TITLE: CATALASE TEST
AIM: to identify gram positive bacteria
METHODS: 1. Slide method
2. Tube method
NB: Report only one method according to the materials provided (slide or tube).
PRINCIPLE: The enzyme catalase acts as a catalyst in the breakdown of hydrogen peroxide to
oxygen and water
2H2O2 Catalase 2H20 + O2
When organisms containing catalase come in contact with hydrogen peroxide, bubbles of
oxygen are given off.
MATERIALS: Test organism, 3% hydrogen peroxide, test tubes, grease free glass slide, cover slip,
applicator stick, test tube rack.
PROCEDURE: (Slide)
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1. Few colonies of the test organism was emulsified in distilled water on a clean grease
free glass slide using a sterile applicator stick.
2. One drop of 3% hydrogen peroxide was added and covered with cover slip
3. The slide was observed for oxygen bubbles.
PROCEDURE: (Tube)
1. 2-4ml of hydrogen peroxide was poured into a test tube.

2. Using a sterile applicator stick, several colonies of the test organism was removed and
immersed in the solution of hydrogen peroxide
3. The test tube was observed for immediate bubbles.
RESULT:
Bubbles observed = catalase positive
Bubbles absent = catalase negative
NOTE:
Do not use your wire loop to pick the colony and touch the 3% hydrogen peroxide. It may give a
false positive result due to its copper nature which can react with the hydrogen peroxide.
CONCLUSION: If catalase positive (the organism is a staphylococcus specie) and if catalase
negative (the organism is a streptococcus specie).
TITLE: COAGULASE TEST

AIM: To differentiate staphylococcus aureus from other staphylococci species
METHODS: 1. Slide method
2. Tube method
NB: Report only one method according to the materials provided (slide or tube)
PRINCIPLE: The free coagulase secreted by staph aureus reacts with coagulase reacting factors
(CRF) in plasma to form a complex called thrombin. This converts fibrinogen to fibrin resulting
in the clotting of plasma
MATERIALS: test colony of the organism, wire loop, clean grease free glass slide, distilled water,
burnsen flame.
PROCEDURE: (slide)
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1. Colony of the test organism was emulsified in a drop of distilled water on a clean grease
free glass slide.
2. A loopfull of plasma was added to the suspension and mixed gently
3. The slide was observed for clumping of the organism within few seconds.
RESULT:
 Clumping of the suspension seen after few seconds = coagulase positive

 Suspension remains uniform after rocking = coagulase negative
NOTE: Call the examiner for initials of all the tests carried out.
CONCLUSION:
If coagulase positive (the organism is staphylococcus aureus) and if coagulase negative (the
organism is other staphylococcus specie).
QUESTION 4
Plate B is a 24hr growth culture plate of urine specimen from a subject with history of urinary
tract infections (UTI). Identify as far as possible the isolates of the plate using the biochemical
reagents provided.
ANSWER
DATE: 25/10/2017
TITLE: IDENTIFICATION OF BACTERIA
AIM: to identify the bacterial organism present in plate labelled B
METHOD 1: Colonial morphology
ON BLOOD AGAR: These are the characteristics of this bacteria

Parameters S. aureus E.coli Kleb proteus pseudo strep
size 0.5mm 2.0mm 3.0mm 2.0mm 2.0mm 0.1mm
colour Cream pale pale pale pale pale
odour NIL NIL NIL Stock fish new NIL
slippers
consistency butyrous mucoid butyrous butyrous butyrous butyrous
opacity
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shape round round round irregular round round

hemolysis
elevation raised raised raised flat flat raised
edge entire entire entire crenated entire crenated
pigmentation NIL NIL NIL NIL green NIL
ON MacConkey
Parameters S.aureus E.coli Kleb proteus pseudo strep
colour Pink pink pink pale green pink
slippers
consistency butyrous butyrous mucoid butyrous butyrous butyrous
opacity
hemolysis
edge entire entire entire crenated crenated entire
on CLED
Parameters S. aureus E.coli Kleb proteus pseudo strep

colour yellow yellow yellow pale green green
slippers
consistency butyrous mucoid butyrous butyrous butyrous butyrous
opacity
hemolysis
edge entire entire entire crenate entire crenate
22
METHOD 2:
PRINCIPLE: see above
Pasteur pipette.
PROCEDURE:
See previous procedure
RESULT:
Report your observation based on e.g
1 colour of gram reaction purple
2 arrangement clusters
3 shape cocci
COMMENT: it was observed that sample C contains gram Positive bacteria

NOTE:
Proceed with biochemical test depending on the organism (gram positive or negative)
METOD 3: BIOCHEMICAL TEST FOR GRAM METOD 3: BIOCHEMICAL TEST FOR GRAM
POSITIVE BACTERIA NEGATIVE BACTERIA
23
TITLE: CATALASE TEST TITLE: OXIDASE TEST

AIM: to identify gram positive bacteria which AIM: To differentiate between gram negative
can produce the enzyme catalase from bacteria which can produce the enzyme
noncatalase producers. oxidase (pseudomonas, Neisseria, vibrio,
METHODS: 1. Slide method brucella and pasturella species) from other
2. Tube method gram negative bacteria which do not produce
NB: Report only one method the enzyme.
PRINCIPLE: When a piece of filter paper is
PRINCIPLE: soaked with a few drops of oxidase reagent
and a colony of the test organism is then
smeared on the filter paper. If the organism
is oxidase producing, the phenylenediamine
in the reagent will be oxidized to deep purple
MATERIALS: Test organism, 3% hydrogen colour.
peroxide, test tubes, grease free glass slide, PROCEDURE:
cover slip, applicator stick, test tube rack
1. A piece of filter paper was placed in a
PROCEDURE: (Slide)
clean petridish, then 2 drops of fresh
1. Few colonies of the test organism prepared oxidase reagent was added
was emulsified in distilled water
2. Using a piece of sterile applicator
on a clean grease free glass slide
stick, a colony of the test organism
using a sterile applicator stick.
was smeared on the filter paper
2. One drop of 3% hydrogen looking out for the development of a
peroxide was added and covered blue purple colour within few
with cover slip seconds.
3. The slide was observed for oxygen RESULT:
bubbles. Blue purple colour observed = oxidase
PROCEDURE: (Tube) positive
1. 3ml of hydrogen peroxide was no blue-purple colour observed = negative
poured into a test tube. oxidase
2. Using a sterile applicator stick, COMMENT: organism on plate B is Oxidase
several colonies of the test positive or negative
organism was removed and
immersed in the solution of
hydrogen peroxide
3. The test tube was observed for
immediate bubbles formation.
RESULT:
Bubbles observed = catalase positive
Bubbles absent = catalase negative
COMMENT:
24
25
TITLE: COAGULASE TEST TITLE: MOTILITY TEST

AIM: To differentiate staphylococcus aureus AIM: to detect the ability of bacteria to move
from other staphylococci METHODS: from one place to another.
1. Slide method METHOD: Hanging drop
2. Tube method MATERIALS: grease free glass slide, cover slip,
overnight broth culture, microscope,
MATERIALS: test colony of the organism, plasticine
wire loop, clean grease free glass slide, PROCEDURE:
distilled water, burnsen flame. 1. Plasticine was used to make a ring of
about 2cm in diameter on a clean
PROCEDURE: (slide) grease free glass slide
1. Colony of the test organism was 2. A drop of a well-mixed overnight
emulsified in a drop of distilled water broth culture was placed on the
on a clean grease free glass slide. center of a coverslip.
2. A loopfull of plasma was added to the 3. The ring of plasticine was carefully
suspension and mixed gently pressed on the coverslip containing
3. The slide was observed for clumping the broth culture.
of the organism within few seconds. 4. It was inverted immediately so that
RESULT: the coverslip was on top.
 Clumping of the suspension seen after 5. It was then examined under the
few seconds = coagulase positive microscope using 10X, then 40X
 Suspension remains uniform after objective respectively with the
rocking = coagulase negative NOTE: condenser low and iris closed.
 Call the examiner for initials of all
the tests carried out. RESULT:
Motility positive (motile organism present)
CONCLUSION: But if you don’t observe motility  motility
If coagulase positive (the specie is staph negative (motile organism absent).
aureus)
if coagulase negative (the specie is other NOTE
staphylococcus specie) Call the supervisor for initials.
CONCLUSION: true motility was seen which

indicate the presence of gram negative bacilli.
26
TITLE: INDOLE TEST

AIM: To assist in the identification of
enterobacteria which are indole productive
(strains of E.coli, P vulgaris, P. rettgeri, M.
morganii and providencia species) which
break down the amino and tryptophan with
the release of indole.
PRINCIPLE: The test organism is cultured in a
medium which contains tryptophan. Indole
production is detected by kovac or ehrlichs
reagent which contains 4(p)-dimethylamino
benzaldehyde. This react with the indole to
produce a red coloured compound.
MATERIALS: peptone water, kovacs reagent,
wireloop, Pasteur pipette, PROCEDURE:
1. The test organism was inoculated in a
bijou bottle containing 3ml of sterile
tryptone water
2. It was incubated at 37oC up to 48hrs
3. Indole production was tested by
adding 0.5ml of kovacs reagent and
then shaken gently.
4. It was examined for a red colour in the
surface layer within 10min. NOTE: if
you’re provided with already
inoculated bottle, start from step 3)
RESULT:
Red surface layer = indole positive yellow
surface layer = indole negative
COMMENT: Organism on plate O is indole
positive or negative.
27
QUESTION 5
Using the materials provide
a. Prepare a nutrient agar plate containing 1/50,000 dilution of rystal violet

b. Subculture one of the organisms on plate G to obtain a discrete colonies using the
quadrant streak method.
c. Briefly explain the significance of using quadrant streak method in (b) above
ANSWER (5a)
TITLE: PREPARATION OF CRYSTAL VIOLET NUTRIENT AGAR
AIM: To cultivate fastidious organisms
MATERIALS: molten nutrient agar, 1% crystal violet (as provided), petridish, water bath, hot air
oven, Pasteur pipette.
Using the formular
R = 1/50,000 V= 20ml O = 1/100

Therefore
= 0.04ml
PROCEDURE:
1. The agar was melted in a hot water bath

2. The agar was allowed to cool to 40-45oC
3. 0.04ml was aseptically removed from the 20ml of the molten agar provided and
discarded.
4. 0.04ml of the 1% crystal violet was added to it
5. It was shaken well and was used to pour into a sterile petri dish
6. It was covered immediately and left on the bench to gel
7. It was then taken into the hot air oven to dry the plate
8. The media was ready for use (or store at 4oc)
28
ANSWER (5b)
TITLE: SPECIMEN SUBCULTURE
AIM: to obtain discrete colonies of the organism growing on plate G
MATERIALS: wireloop, burnsen burner, nutrient agar containing 1/50,000 dilution of crystal
violet, incubator, plate labelled G.
METHOD: streaking
PROCEDURE:
1. The agar plate was dried in the hot air oven to reduce the moisture
2. A wireloop was sterilized in a flame of burnsen burner and allowed to cool properly.
3. Molten nutrient agar was poured into a petridish and allowed to set on cooling while
drying at the same time (If not already made).
4. The sterilized wireloop was used to pick a colony of the test organism.
5. The organism was inoculated into the nutrient agar (containing 1/50,000 dilution of
crystal violet) and was spread by means of streaking in a unique manner.
6. The sample was incubated at 37oC for 24hrs.
NB :( do not report step 6 if you are asked to leave on the bench)
DIAGRAM:
ANSWER (5c)
This method is used most commonly to isolate pure cultures of bacteria. A small amount of
mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the
surface of the agar medium. The successive streaks “thin out” the inoculum sufficiently and the
micro-organisms are separated from each other.
It is usually advisable to streak out a second plate by the same loop/needle without
reinoculation. These plates are incubated to allow the growth of colonies. The key principle of
this method is that, by streaking, a dilution gradient is established across the face of the Petri
plate as bacterial cells are deposited on the agar surface.
Because of this dilution gradient, confluent growth does not take place on that part of the
medium where few bacterial cells are deposited. Presumably, each colony is the progeny of a
single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked
up separately using sterile inoculating loop/needle and re-streaked onto fresh media to ensure
purity
29
QUESTION 6
With the materials provided prepare and pour a 10% blood agar. Streak the plate so prepared
to obtain a discrete colony.
ANSWER (6)
TITLE: PREPARATION OF BLOOD AGAR
AIM: To cultivate fastidious organisms
MATERIALS: As provided
𝑹𝑽
Using the formular
𝑶
R = 10% V = 20ml O = 100%
2ml
PROCEDURE:
1. 2ml of the molten media was withdrawn and discarded aseptically

2. 2ml of blood provided was added aseptically and was mixed well
3. The media was poured into a sterile labelled petridish
4. It was covered immediately and allowed to gel
5. The plate was
6. Dried in the hot air oven to reduce the surface moisture
7. The plate was brought out for streaking
TITLE: PLATING/CULTURING
AIM: to obtain discrete colonies of the organism growing on the given plate/specimen
MATERIALS: wireloop, burnsen burner, molten media, incubator, sample plate.
METHOD: streaking
PROCEDURE:
30
5. The organism was inoculated into the blood agar and was spread by means of streaking
in a unique manner.
6. The sample was incubated at 37oC for 24hrs
NB :( do not report step 6 if you are asked to leave on the bench or report: it was left on the
bench)
DIAGRAM: see diagram above
QUESTION 7
With the materials provided, prepare and pour a 5% blood agar plate containing 1/250,000
dilution of crystal violet and subculture pure of the organisms on the plate to obtain discrete
colonies (2% crystal violet is provided)
ANSWER (7)
TITLE: PREPARATION OF 5% BLOOD AGAR PLATE
AIM: To prepare and pour a 5% blood agar plate containing 1/250,000 dilution of crystal violet
and subculture pure of the organisms on the plate to obtain discrete colonies
MATERIALS: petri dish, hot air oven, petridish, pipette
For 5% blood agar
1ml
For 1/250,000 crystal violet
0.004ml
= 1ml of Blood + 0.004ml of crystal violet
= 1.004ml
PROCEDURE:
1. 1.004ml of molten media provided was withdrawn aseptically and discarded

2. 1ml of blood provided and 0.004ml of 2% crystal violet were added aseptically and
mixed well.
31
3. It was pour into a clean sterile labelled petri dish

4. It was allowed to gel, and was dried in hot air oven and ready for streaking
QUESTION 8
a. With the material provided, prepare and pour a chocolate agar containing 2% blood,
and subculture the organism on the plate to obtain a discrete colony.
b. Perform a tube test to determine the pathogenicity of any selected group and report
your findings accordingly.
ANSWER (8a)
TITLE: PREPARATION OF CHOCOLATE AGAR PLATE CONTAING 2% BLOOD
AIM: To prepare and pour a chocolate agar containing 2% blood and subculture pure of the
organisms on the plate to obtain discrete colonies
MATERIALS: petri dish, hot air oven, petridish, pipette, 2% blood
R = 2% of blood provided V = 20ml O = 100%
= 0.4ml
PROCEDURE:
1. The molten medium was allowed to cool to about 50oC and 0.4ml was withdrawn and
discarded.
2. 0.4ml of the blood provided was added to it and was mixed well
3. The mixture was taken back to the water bath and warmed to the temperature of 80oC
and then it was removed
4. It was then poured into a clean sterile labelled petridish and allowed to gel.
5. Plate was dried using hot air oven and ready for subculturing
TITLE: SPECIMEN SUBCULTURE

AIM: to obtain discrete colonies of the organism growing
MATERIALS: wireloop, burnsen burner, nutrient agar containing 1/50,000 dilution of crystal
violet, incubator, plate labelled G.
32
METHOD: streaking
PROCEDURE:
5. The organism was inoculated into the nutrient agar (containing 1/50,000 dilution of
crystal violet) and was spread by means of streaking in a unique manner.
6. The sample was incubated at 37oC for 24hrs (don’t write this if you are asked to leave on
the bench)
ANSWER (8b)
The test to be carried out is coagulase test. Ask for the overnight cultures of the organism in
nutrient broth. If not provided, then prepare a suspension of the organism in broth.
QUESTION 9
Carryout a macroscopic examination of sample marked A and comment on your findings.
Make films and stain one by gram method and the other by Zn staining. Examine and
comment on your findings.
ANSWER (9)
GIVEN: Sputum sample marked A
MACROSCOPY
Appearance = blood stain
Constituent = Nil
Consistency = mucoid
33
See gram staining above
TITLE: ZN STAINING
AIM: to demonstrate the presence of acid-fast bacilli using zeil-nelson stain
PRINCIPLE: Members of the bacterial genus Mycobacterium contain large amount of lipids
(fatty) substances within their cell walls. These fatty waxes resist staining by ordinary methods.
When these organisms are stained with a concentrated solution of a basic dye such as carbol
fuchsin, while applying heat, the stain can penetrate the lipid cell wall and reach the cell
cytoplasm. Once the cytoplasm is stained it resists decolourization even with harsh agents such
as acid alcohol which cannot dissolve and penetrate beneath the mycobacterial lipid wall.
Under these conditions of staining the mycobacteria are said to be acid fast. Other bacteria of
those, cell walls do not contain high concentration of lipid are readily decolorized by acid
alcohol after staining with carbol fuchsin and are said to be nonacid fast
PROCEDURE:
1. A Smear was made and heat fixed

2. The slide was flooded with carbol fuchsin and was heated gently until stream rises and
stain for 5min
3. The stain was rinsed with tap water
4. It was decolorize with 20% H2SO4 for about 5min
5. It was washed in tap water
6. It was counter stained with 0.5% methylene blue for 1min
7. It was Rinsed with distilled water
8. It was blotted, dried and examine under oil immersion
RESULT:
AFB Bacteria……..pinkish-red
Background of other component……blue
34
STUDY QUESTIONS
1 a. Broth labelled M is an overnight broth culture of a wound swab
i. Identify the medium and carryout gram staining on the broth and report
your findings. ii. Subculture the broth on the nutrient agar plate provided,
incubate your plate and present it at the VIVA session.
b. Plate N is an overnight culture of the broth labelled M
i. Describe the morphological features of the colonies present ii.
Identify the organism present as far as possible using the material
provided.
2 The plate labelled A is an overnight culture of an aspirate.

a. Gram stain the colony growing on the plate and report your findings
b. Perform the following biochemical test
i. Catalase ii.
coagulase
3 sample G is a urine sample collected from a 25year old male subject who presented
with a history of UTI in AKTH
a. centrifuge the urine sample labelled G, examine the deposit and report
your findings
b. State the clinical significance of your findings from the deposit.
4 a. Examine the deposit of the urine marked C and record your findings,
plate/culture the sample on the MacConkey agar provided: leave on the bench
b. The plate marked D is a mixed culture, carry out routine test on it as follows.
i. Describe the colonial appearance of the organism ii. Make
film stained by gram method and comment on the results iii. With the
materials provided, determine whether or not some of the organisms
produce pathogenic enzymes
35
HEMATOLOGY & BGS

QUESTION 1
a. With the materials provided, determine the ABO blood group of the samples marked
D,E,F and G
b. Carryout a direct coombs test on serum sample H and comment on your result.
ANSWER 1(a)
DATE: 30/09/2018
TITLE: ABO and RHESUS BLOOD GROUPING
AIM: To carryout cell grouping on the given sample
MATERIALS: Pasteur pipette, applicator stick, cotton wool, antisera A, B and D
METHOD: Tile method
PRINCIPLE: Antigen + antibody Agglutination
PROCEDURE:
1. The given sample was washed three (3) times with normal saline and 20% cell
suspension was made (1drop of cell + 19 drop of normal saline).
2. The tile was clean and dried and a drop of antisera A, B and D were placed onto
different column
3. A drop of 20% cell suspension of the cell were added on the antisera respectively.
4. They were mixed using the applicator stick
5. The tile was rocked for 5min (for antigen antibody to react) RESULT:
SAMPLES ANTI-A ANTI-B ANTI-D REMARK
D + - - A Rhesus D Negative
E - + + B Rhesus D positive
F - - + O Rhesus D positive
G + + + AB Rhesus D positive
36
(+) = agglutination (-) = no agglutination

CONCLUSION: The various blood groups of the samples were determined.
 It is a screening test for blood donation

 It is a screening test for blood transfusion
ANSWER 1(b)
TITLE: DIRECT COOMBS TEST
AIM: To determine the in vivo sensitization of the given blood sample
MATERIALS: AHG, washed blood sample, test tubes, glass slides.
METHOD: Tube method
PRINCIPLE: Direct antiglobulin test is applied to detect red cells coated in vivo with
immunoglobulin or compliment. Washed red cells from donor is tested directly with antihuman
globulin reagent. The test is used to detect hemolytic disease of the new born, autoimmune
hemolytic anemia, sensitization of red cells due to drugs
PROCEDURE:
1. The patients red cells were washed three (3) times with saline
2. The final supernatant was discarded and the concentrated cells were used to make 5%
cell suspension
3. One drop of cell suspension was added into a tube and another drop was added into
another tube
4. Equal volume of AHG was added into one of the tubes and a drop of saline to the other
tube to serve as control
5. The mixtures were mixed with a stick and allowed to stand for 1minute. At 30 sec
interval, each tube was packed up and tapped to encourage agglutination
6. The mixtures were spun lightly for 15 sec at 1000rev/min
7. The tubes were observed for the presence of agglutination
If there is still no agglutination, add 1 drop of igG coated red cells to any cell that is negative.
Mix and centrifuge for 1500rev/min for 1min, if there is no agglutination, the test result is
invalid and the test should be repeated. If agglutination is obtained, test is valid.
RESULT
TEST TUBES RESULT
H Positive(+)
37
positive control Positive(+)

negative control negative (-)
COMMENT:
QUESTION 2
a. With the materials provided, determine the ABO blood group of the Sera marked P, Q,
R and S
b. Carryout an indirect Anti Human Globulin Test (AHG) on serum sample H and
comment on your result.
Answer (2a)
DATE: 25/10/2017
TITLE: SERUM GROUPING
AIM: To carryout Serum grouping on the given sample P, Q, R and S
MATERIALS: Pasteur pipette, applicator stick, cotton wool, pooled cells A, B, O, White tile,
METHOD: Tile method
PROCEDURE:
1. The tile was clean and dried and a drop of sera P, Q, R and S were placed onto different
column
2. A drop of Pooled red cells were added on the sera respectively.
3. They were mixed using the applicator stick
4. The tile was rocked for 5min (for antigen antibody to react)
5. Agglutination was observed.
RESULT:
SAMPLES/SERA POOLED CELL POOLED CELL B POOLED CELL O REMARK
A
P - - - AB
Q - + - A
R + - - B
S + + - O
38
(+) = agglutination (-) = no agglutination

Verify the interpretation please
CONCLUSION: The various blood groups of the samples were determined.
 It is a screening test for blood donation

 It is a screening test for blood transfusion
ANSWER 2 (b)
DATE: 29/9/2017
TITLE: INDIRECT COOMBS TEST

AIM: To detect the presence of invitro sensitization of the given sample.
METHOD: Tube method
PRINCIPE: The indirect Antiglobulin is done to determine the sensitization of RBC with igG or
compliment invitro.
MATERIALS:
PROCEDURE:
1. Three (3) drops of serum were added on a test tube

2. One drop of 5% suspension of washed red cells of O Rh D positive patient was added to
the serum in the test tube
3. The mixture in the test tube was mixed and incubated at 37oC for 40min
4. The mixture was centrifuged for 1min at 1000 rev/min
5. The mixture was observed for hemolysis or agglutination RESULT:
SAMPLES RESULT
H +
CONCLUSION:
QUESTION 3
a. Carryout the one stage Prothrombin time test on the control plasma J and samples
marked K and L and calculate the following: i. Prothrombin time ii.
Prothrombin ratio iii. Prothrombin index iv. INR using ISI value
of 1.3
39
b. Determine the reticulocytes count of the sequestered blood sample marked M and
comment on your finding.
ANSWER (3a)
DATE: 23/7/2019
TITLE: PROTHROMBIN TIME TEST
AIM: to determine prothrombin time test of plasma sample marked K and L
GIVEN: plasma sample marked K and L
METHOD: Prothrombin time (quick one stage method)
PRINCIPLE:
MATERIALS: Test tubes, water bath, stop watch,
PROCEDURE:
1. Three (3) clean tubes were labelled J, K and L were arranged in the water bath at
temperature 37oC
2. The working reagent and the sample was pre warmed at 37oC for few seconds
3. The working reagent (CaCl + thromboplastin) was mixed gently and 200µL (0.2ml) was
pipetted into the test tube and incubated for 5min at 37oC
4. 100µL (0.1ml) of sample k and L and control J was then dispensed into the tubes.
5. The stop watch was switched on immediately
6. The tubes were agitated continuously inside the water bath and observed at few
seconds interval
7. The stop watch was stopped as soon as clotting was noted
8. These were repeated twice to take the average of the test
RESULT:
SAMPLES first reading second reading average
K 13 15 14
L 15 17 16
J(control) 12 12 12
40
CALCULATIONS:
i. Prothrombin time
Sample K = 14sec
Sample L = 16sec
ii. Prothrombin ratio

Ratio of test to control
For sample K = 14:12 = 7:6
For sample L = 16:12 = 4:3
iii. Prothrombin index
Prothrombin index (control to test) =
For sample K =
For sample L = iv.
INR
INR ISI
INR = International normalized ratio
ISI = international sensitivity index
Therefore
INR (sample K) = [
INR (sample L) = [
41
NORMAL RANGE = 11-18sec

COMMENT:
PT or Quick test has remained an important test for detecting disorders of blood coagulation. It
is the most common coagulation procedure performed in routine lab apart from APTT. The PT is
particularly sensitive to defects of the extrinsic coagulation pathway (factor 2, 5, 7, 10 and
fibrinogen as well as its inhibitors. It is an indicator of hepatic disease. It is also the most
common used test for monitoring oral anticoagulant therapy. PT is commonly used for
monitoring heparin anticoagulant therapy.
QUESTION 4
a. Determine the ABO and Rh(D) blood group of the blood samples marked F, G, H, I and
J
b. Use the result in (a) above to determine the optimum working titre of the serum
marked K.
ANSWER (4b)
DATE: 24/10/2019
TITLE: ANTIBODY TITRATION
AIM: To determine the optimum working titre of the serum marked K
MATERIALS: eleven (11) test tubes, microscope, normal saline, pipette, 5% suspension of
standard cells.
PROCEDURE:
1. Eleven test tubes were arranged serially from one to eleven (1-11)
2. Two drops of normal saline was added in each of the test tube excluding test tube 1
3. Two drops of serum was added in test tube 1 and 2
42
4. The contents of test tube 1 and 2 were mixed gently and 2 drops from the content of
test tube 2 was transferred into test tube 3 and so on until the last test tube was
reached
5. The last two drops was discarded and two drops of 5% cell suspension of the
corresponding cells was added
6. The test was incubated for 1hr 30min at room temperature
7. A drop of mobile albumin as a catalyst was added and incubate further for 30min
8. The result was read macro and microscopically for the presence of agglutination
RESULT:
Vol of test tubes 1 2 3 4 5 6 7 8 9 10 11

Volume of NS - 2 2 2 2 2 2 2 2 2 2
Volume of serum 2 2 2 2 2 2 2 2 2 2 2
Volume of 5% 2 2 2 2 2 2 2 2 2 2 2
cells
Total volume 4 4 4 4 4 4 4 4 4 4 4
Concentration clear 1
1024
Result +++ +++ +++ ++ ++ + - - - - -
CONCLUSION:
From the above result obtained, the serum belong to group B (anti A). A good serum should
have a titre of 1/128 against A cells and 1/64 against A2 cells but the serum shows highest
agglutination hence cannot be used as a diagnostic serum antisera.
The antibody titre value of the experiment was found to be 1/32 in the exact test tube were last
agglutination is seen.
QUESTION 5
a. Determine the reticulocyte population on the sequestered blood sample marked P
and report your result in reticulocyte percent. Comment on the result
43
b. Determine the parked cell volume (PCV) of samples Q and R and record your result in
L/L
ANSWER 5(a)
DATE: 23/03/2017
TITLE: RETICULOCYTE COUNT
AIM: To count the percentage of immature red blood cells in the given sample.
METHOD: Manual method
PRINCIPLE: an isotonic solution of supravital stain concentration that stains living materials eg
new methylene blue or brilliant cresyl blue is incubated with few drops of blood to detect
ribosomal RNA in reticulocyte. The red blood cell must be stain while they are still living. A thin
film is made and the reticulocyte is counted microscopically. Reticulocyte are recognised by the
violet stain granules or ribosomal RNA called reticulin. The reticulocyte is expressed as
percentage and preferably in absolute number when RBC is available,
MATERIALS: microscope, glass slide, water bath, cover slip, test tube, oil immersion, new
methylene blue
PROCEDURE:
1. 2-3 drops of the stain solution was placed in a test tube.

2. Equal drop of patient EDTA blood sample was added to the test tube and mixed.
3. The mixture was incubated at 37oC for 15min
4. The cells were suspended in a test tube
5. A small drop of the mixture was placed on the slide and a thin film was made and The
film was allowed to air-dry
6. The reticulocyte was counted using the immersion oil objective RESULT:
NUMBER OF RED BLOOD CELLS NO OF RETICULOCYTE
350 1
200 3
251 4
174 2
975 10
CALCULATION:
44
Reticulocyte % X 100%
x 100%
= 1.03%
Normal range
Adult = 0.2 – 2%
Infant = 2-5%
CONCLUSION:
The reticulocyte percentage was found to be 1.03% which falls within the normal range
therefore the patients process of Haemopoiesis is functioning well
COMMENT:
The subject from which this sample was collected was not suffering from hypoplastic bone
marrow or hemolysis
ANSWER (5b)
DATE: 2/2/2020
TITLE: PACK CELL VOLUME
AIM: To determine the relative mass of erythrocyte or packed cells present in the given sample.
METHOD: Microhaematocrit method
PRINCIPLE: A sample of whole blood (anticoagulated is centrifuge for 12,000-15,000 revolution
per minute for 3-5min to achieve maximum packing of the cell and plasma.
MATERIALS: hematocrit centrifuge, hematocrit reader, plasticine, capillary tubes (heparinized
or non-heparinized)
PROCEDURE:
1. 2/3 of the plain capillary tube was filled with well mixed anticoagulated blood
2. The unused capillary tube end was sealed with plasticine
3. The filled capillary was placed in one of the number lot of the hematocrit rotor with the
seal end around the lid gasket
4. The blood was centrifuge for 15,000 rev per minute for 3-5 min
5. The packed cell volume was read using hematocrit reader RESULT:
Samples PCV (L/L)
Sample P 0.39
45
sample Q 0.44
NORMAL RANGE: With regard to age and sex

Men = 0.40 – 0.54 L/L
Women = 0.36 – 0.46 L/L
6 – 12 yrs = 0.35 – 0.45 L/L
2-5 yrs = 0.34 – 0.40 L/L
Infants = 0.44 0.54 L/L
COMMENT: The PCV of the subjects labeled P and Q is within the normal range
 To screen anemia or polycythemia

PRECAUTION
 I ensured that the sample was well mixed before used
 I ensured that the sample was not overspun
QUESTION 6
a. Determine the PCV of the blood sample X and report your result in S.I unit
b. You are provided with certain hematological parameters of sample X above as follows
[Hb = 13g/dl RBC = 5 x 1012/L ] calculate the following
i. MCV ii.
MCH
iii. MCHC and comment on your result
ANSWER (6b)
GIVEN Hb = 13g/dl RBC = 5 x 1012/L
PCV was found to be 39% = 39/100 =0.39 L/L i.
MCV = 7.8 x 10-14

Normal range for MCV =
46
ii. MCH = = 2.4 x 10-10

Normal range for MCH = 27-32pg
iii. MCHC = 213g/L

Normal range for MCHC = 315 -360 g/L Normal range = 32 – 35 g/L
QUESTION 7
a. Determine the ABO and Rh blood group of samples marked K, L,M and N
b. Use the reagents provided to carryout sickling test on blood sample marked E and F
and report your findings
ANSWER (7b)
TITLE: SICKLING TEST
AIM: To determine the presence or absence of sickle cell in the given sample.
METHOD:
MATERIALS: 2% sodium metabisulphide, Normal saline, test tubes, microscope slide, plasticine
or Vaseline, incubator, microscope.
PRINCIPLE: Red blood cell containing hemoglobin S will sickle when expose to a strong reducing
agent, such as sodium metabisulphide which is a convenient reducing agent for screening of
HbS. Whole blood cell is mix with freshly prepared sodium metabisulphide and a wet solution is
examine microscopically for the presence or absence of sickle cell.
PROCEDURE:
1. 1 in 5 dilution of blood was made by adding 4 drops of saline to 1 drop of blood 2.

A small drop of the diluted blood was added in a separate test tube.
3. Equal volume of 2% sodium metabisulphide was added and then mixed well
4. On a clean microscope slide, a ring of Vaseline was made
5. A small drop of the mixture above was added in the space circle by the Vaseline
6. A cover slip was applied, pressed slightly onto the Vaseline ring
7. The preparation was incubated in a wet chamber for 10min
8. The slide was examined using 10X and 40X objective to determine the presence of sickle
cell
RESULT:
47
Sample A = positive (sickle cell found) Sample

B = negative (no sickle cell found)
Note:
HbAs = 30 – 50% of red cells sickled
HbAA = no sickle cell found
COMMENT:
In sample A, sickle cells and normal cell were observed while all the cells in given sample were
observed to appear normal.
QUESTION 8
Sample M is from a patient with PUD. Carry Out Hb estimation on sample labelled M using J
as your standard (12.0g/dL). Calculate the MCHC of the sample labelled M and comment on
your findings.
ANSWER 8
DATE: 13/5/2017
TITLE: HEMOGLOBIN ESTIMATION
AIM: To estimate the hemoglobin concentration of the given blood sample
METHOD: cyanmethaemoglobin method
PRINCIPLE: potassium ferricyanide convert the haemoglobin to methaemoglobin which is
further converted to cyanmethaemoglobin.
Hb + potassium ferricyanate  methaemoglobin
Methaemoglobin + potassium cyanate  cyanmethaemoglobin
The colour intensity produced is proportional to the concentration of hemoglobin in the blood.
This colour intensity is measured at 540nm with a spectrophotometer/colorimeter
48
MATERIAL: spectrophotometer, distilled water (1000ml), potassium dihydrogen phosphate

(140mg), potassium cyanide (50mg), potassium ferricyanide (200mg), reagent (Drabkins
solution), pipette.
PROCEDURE:
1. 4ml of Drabkins solution was pipetted and dispensed into clean test tube
2. 0.02ml of the well mixed sequestrated blood was pipetted and was dispensed into it and
mixed well
3. The above steps where repeated with blank and standard
4. It was allowed to stand for 10minutes for complete conversion
5. It was read spectrophotometrically against the reagent blank of 540nm
PROTOCOL: the test tubes were arranged as follows
TEST STANDARD BLANK
sample 0.02ml - -
standard - 0.02ml -
Drabkins 4ml 4ml 4ml
NOTE:
ASK for the haemoglobin standard and its concentration
RESULT:
Concentration of the given haemoglobin standard = 57.2 mg/dL
Dilution factor = 251 (5ml) or 201 (4ml)
Optical density of the STD = 0.46
Optical density of the test = 0.39
CALCULATION:
Haemoglobin estimation = ] X DF
NOTE: If the conc of STD is in mg/dL. You have to divide the conc of STD by 1000, but if it is only
given in e.g 0.0572, then don’t divide the conc of STD by 1000.
Haemoglobin estimation = X DF
HENCE
Haemoglobin estimation = X DF
49
x 251 = 12.17mg/Dl
Hb = 12.17mg/dL 12.17g/L
MCHC = = 32.89 g/L
Normal range = 32 – 35 g/L
NORMAL RANGE:
Male = 13.5 – 18.0
Female = 11.5 – 16.5
Infants = 13.5 – 19.5 Children
= 9.5 – 12.5
COMMENT:
If the Hb concentration is less than the normal range, the subject is anemic (or if > 20g/dl
subject is polycythemic).
This results fall in between the normal range. This means that the patients’ blood estimation is
still normal.
The further test I will suggest for the patient are
 Osmotic fragility test

 Differential count
 Red blood cell count
 White blood cell count CLINICAL SIGNIFICANCE:
 To confirm anemia
 To monitor the response of anemic subject to treatment
 To confirm polycythemia
QUESTION 9
Prepare 4 tubes each containing 5ml amount of doubling dilution of NaCl solution marked J.
to each tube add 0.02ml of fresh formed blood marked K and mix, stand for 30minutes and
centrifuge. Estimate colorimetrically at 540nm the percentage of haemolysis in each
supernatant fluid from your results.
50
ANSWER (9)
DATE: 30/12/2018
TITLE: OSMOTIC FRAGILITY TEST
AIM: To determine the percentage of haemolysis in blood sample
GIVEN: Sample marked K METHOD:
Osmotic fragility test
PROCEDURE:
The test was carried out using the protocol below
No of tube 1 2 3 4
vol of D/water (ml) 5 5 5 5 discarded
vol of NaCl (ml) 5 5 5 5
dilution factor ½ 1/4 1/8 1/16
concentration 0.5 0.25 0.125 0.06
vol of blood sample K 0.02 0.02 0.02 0.02
(cm3)
 It was then mixed well and incubated at room temperature for 30min
 It was then centrifuged and was read colorimetrically at 540nm
optical density of 0.1 0.2 0.3 0.4

samples
% lysis 25 50 75 100
CALCULATIONS:
% lysis =
NOTE:
The OD of STD is the tube that has none or minimum amount of NaCl
Tube 1 = x 100 = 25%
Tube 2 = x 100 = 50%
Tube 3 = = x 100 = 75%
51
Tube 4 = = x 100 = 100%
QUESTION 10
You are provided with 1% buffered sodium chloride labelled L. dilute with distilled water to
give concentration % 0.1, 0.2, 0.3, 0.4, 0.45. 0.55, 0.6, 0.7, 0.9. Total volume = 5cm3. Add
0.02cm of blood marked L mix well. Allow to stand on the bench for 30minutes. Centrifuge
and read supernatant haemolysis and plot result on graph sheet provided, comment briefly
on the result.
ANSWER (9)
TITLE: OSMOTIC FRAGILITY TEST
AIM: To know what concentration the cell will lyse
METHOD: spectrophotometer
PRINCIPLE: normal red cell suspended in an isotonic solution or saline (0.89%) remain intact as
the concentration increase or decrease, the cells are disrupted causing haemolysis. The lysis
depends on the shape of the red cells. Normal red cells shows hemolysis at salt concentration
of 0.05% NaCL while sickle cells and spherocytes lyses at lower conc (because of reduced
surface area volume ratio) of 0.6%
MATERIALS:
PROCEDURE:
1. 11 test tubes were arranged in test tube rack
2. 1% working solution was prepared from the stock solution of 10% NaCl by taking 1ml of
a given 10% NaCL to 9mls of distilled water
3. Serial dilution of NaCl was prepared in increasing order of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.85, and 1.0 in 5ml quantity (to each tube in order to make the total volume in
each tube 5ml)
4. 0.02ml of freshly collected heparinized blood was added. Separately to each test tube, it
was then mixed well by inversion and allowed to stand for 30 minutes at room
temperature
5. The tubes were centrifuged at 1500rpm for 5min
6. The supernatant was carefully removed and its optical density was measured at 540nm
7. The first tube was taken as blank (0% lysis) and the last tube as 100% (complete lysis).
1 2 3 4 5 6 7 8 9 10 11
conc of 10% NaCl 0.1 0.2 0.3 0.4 0.45 0.5 0.6 0.7 0.8 0.85 1.0
52
vol of 1% NaCl 0.5 1.0 1.5 2.0 2.25 2.5 3.0 3.5 4.0 4.25 5.0
vol of distilled water 4.5 4.0 3.5 3.0 2.75 2.5 2.0 1.5 1.0 0.75 0.0
Total volume 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
volume of blood 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
concentration/ OD 0.04 0.07 0.06 0.07 0.06 0.07 0.24 0.31 0.19 0.15 0.25
%lysis
NOTE:  To calculate the volume of diluent and 1% NaCL

Diluent = distilled water
= 0.5ml Diluent = 5ml – 0.5 = 4.5ml

Tube 1 =
= 1ml Diluent = 5ml – 1 =4ml
Tube 2 =
= 1.5ml Diluent = 5ml – 1.5 =3.5ml
Tube 3 =
= 2ml Diluent = 5ml – 2 = 3ml
Tube 4 =
= 2.25ml Diluent = 5ml – 2.25 = 2.75ml
Tube 5 =
= 2.5ml Diluent = 5ml – 2.5 = 2.5ml
Tube 6 =
Tube 7 =
= 3.5ml Diluent = 5ml – 3.5 = 1.5ml
Tube 8 =
= 4.25ml Diluent = 5ml – 4.25 = 0.75ml
Tube 9 =
Tube 10 =
NOTE:
OD of standard is the tube with 1% NaCl. The % lysis is used to plot graph with the
concentration of 1% NaCl horizontally. At 50% lysis trace to touch the line of the graph and use
it to trace down to the conc of 1% NaCl.
CALCULATION
% lysis =
53
QUESTION 11
You are provided with blood sample D, perform a total white cell count
ANSWER (10)
TITLE: TOTAL WHITE BLOOD CELL COUNT
AIM: To estimate the total number of white blood cells in blood sample D.
METHOD:
PRINCIPLE: Whole blood is diluted with diluting fluid (Turks solution) which hemolysis mature
erythrocytes and facilitates leukocytes counting.
MATERIALS: blood sample, diluting fluid, improved neubeaur chamber, cover slip, Turks
solution
PROCEDURE:
1. 0.38ml of diluting fluid was dispensed into a test tube

2. 0.02ml of well mixed EDTA anticoagulated blood was added into the test tube and
mixed to make 1:20 dilution
3. The counting chamber was assembled by sliding the cover slip in to position over the
grid areas and pressing it down on each slide until newton’s ring is formed
4. The diluted blood sample was remixed using a Pasteur pipette and one of the grid area
of chamber was filled with the dilution
5. The chamber was placed in a moist petridish
6. The outside of the chamber was allowed to dry and it was placed on the microscope
stage
7. The WBCs were viewed and counted using 10x objectives with condenser iris sufficiently
closed
RESULT
S/N SQUARES NO OF CELLS COUNTED
A 49
B 45
C 55
D 39
total 188
CALCULATION
54
WBC = x DF x DF
Dilution factor = 200
Area of the counting chamber = 4mm2
Depth of the counting chamber = 1/10mm

Volume of the four large squares = 4 x 1/10 = 2/5mm3
Total no of cells from the 4 squares = 188
WBC = x DF = = 94,000 cells/mm3

Convert to S.I unit
94,000 cells/mm3 x106 = 9.4 x 1010/L
STUDY QUESTIONS
1 a. Perform one stage Prothrombin time test on the test and control plasma
samples marked K and L respectively. Express your answer in: i.
Prothrombin time ii. Prothrombin ratio iii. Prothrombin index
iv. INR using ISI value of 1.3
2 a. Blood samples marked D, E, F, G and H are from prospective donors. Determine

the ABO blood groups and Rh factor of the blood samples
b. Carryout an indirect Coombs test on serum sample I suspected to be carrying
clinical significant antibodies. Comment on your result.
3 Blood samples W and X are putative fathers while Y is from the mother and sample Z is
from the child. With the materials provided determine who the likely father of the
child is. What other tests will you perform to establish your findings?
HISTOPATHOLOGY
QUESTION 1
You are provided with a paraffin tissue section X. carryout the following
55
a. Remove the wax completely and carryout all the preliminary treatments until the
section is ready for staining.
b. Stain the section to demonstrate the general tissue structure.
ANSWER (1a)
DATE: 25/10/2017
TITLE: PREPARATORY TREATMENT OF PARAFFIN EMBEDED TISSUE SECTIONS
GIVEN: Tissue section X
REQUIRED TO: Remove the paraffin wax completely and carryout all the preliminary treatments
until the given section is ready for staining.
MATERIALS: clean dry grease free slide, cover slip, immersion oil, xylene, burnsen flame,
absolute alcohol, descending grade of Alcohol.
PROCEDURE:
1. Paraffin wax was removed from the tissue by application of heat and subsequent
treatment in 2-3 changes of xylene for 2mins
2. Xylene was removed by treatment with absolute alcohol, then with descending grade of
alcohol, (absolute 90%, 80% and 70%) and was taken to water.
3. The tissue section was stained
4. The stained section was dehydrated using ascending grade of alcohol (70, 80, 90,
absolute), cleared in xylene and mounted
CONCLUSION: At the end of the practical a paraffin embedded tissue was successfully mounted.
ANSWER (1b)
TITLE: DEMONSTRATION OF GENERAL TISSUE STRUCTURES
GIVEN: paraffin tissue section X
REQUIRED TO: demonstrate general tissue structure
METHOD: Hematoxylin and Eosin
MATERIALS: as provided
PROCEDURE:
1. Tissue section was dewaxed using 2 changes of xylene for 2min

2. Tissue section was hydrated using descending grade of alcohol (100%, 95%, 90%, 70%)
for 2min
3. Tissue was rinsed in distilled water
56
4. Tissue section was stained using hematoxylin (Harris = 5min, Erhlich = 20min, Cole =
10min)
5. Tissue was rinsed in distilled water
6. Tissue section was differentiated using 1% acid alcohol for few seconds
7. Tissue section was rinsed with Scott tap water for 3minutes, or tissue section was rinsed
in tap water for 10minutes
8. Tissue section was counter stain using 1% aqueous Eosin for 3minutes
9. Tissue section was rinsed in distilled water
10. Tissue section was dehydrated using alcohol in ascending grade (70% 90%, 95%, 100%)
for 2min
11. Tissue section was cleared using xylene at 3 changes
12. Tissue section was mounted with DPX and examined under the microscope using 10X,
then 4X objective
RESULT:
 Nucleus…….blue black
 Cytoplasm…….pink
 RBC…….red
 Add the structure that you can see
QUESTION 2
A freshly removed bloody liver biopsy was fixed in acid-formalin for 14 days and processed to
obtain sections X & Y.
a. Stain X to demonstrate general tissue structures

b. Y to complete Tirmann-schmeltzer for ferrous iron (slide already incubated for 3 hours
in ammonium sulphide)
ANSWER (2a)
DATE: 21/03/2017
STATEMENT: Prolonged fixation in acid-formalin result to the formation of formalin pigment.
(Formalin pigment results when acidic formalin solutions react with blood rich tissues such as
spleen and areas of hemorrhage, forming brown or brownish-black crystalline substances that
are bi-refringent.) Hence tissue should be treated to remove the pigment
57
TITLE: REMOVAL OF FORMALIN PIGMENT

GIVEN: Section X & Y
REQUIRED TO: remove formalin Pigment
METHOD: depends on the reagent given
1. Saturated alcoholic picric acid method (S.A.P.A),

2. Alcohol ammonia solution
METHOD 1: saturated alcoholic picric acid method
PRINCIPLE:
PROCEDURE:
1. Tissue section was dewaxed using 2 changes of xylene for 2min

2. Tissue section was transferred to absolute alcohol (100%)
3. Sections were treated with Saturated Alcoholic Picric Acid for 20 minutes (for fine
granules) or very occasional 2hrs (for heavy ppt) [report time depending on sample
given]
4. Sections were taken to water (90%, 70% alcohol) and washed briefly.
RESULT
 Formalin and malaria pigment…………………. Removed
NB: then, the section is ready for staining
METHOD 2: Alcohol ammonia solution (95 % alcohol = 50 mL, Concentrated ammonia (0.880) =
15 ml Mix before use).
PRINCIPLE: Formalin pigment may be present in tissue after long-term fixation in formalin with
an acid pH. It is brown and usually extracellular, but may be present in macrophages. Short
term fixation in neutral buffered formalin may prevent this artefact. It is easily removed with
alcohol - ammonia solution.
PROCEDURE:
1. The section was deparaffinize and hydrate with distilled water.

2. It was Placed in Alcohol-ammonia solution for 1 (one) hour.
3. It was Wash in water.
4. It was Stained using H&E (or mention any other technique used).
58
NOTE:
 Most formalin pigment will be removed fairly rapidly, and the time given should be
adequate.
 Heavy deposits may take longer. Sections may be left overnight if necessary
 Treatment with lithium carbonate is only required if picric acid discolouration interferes
with staining.
NOTE: Report only one of the methods for which the reagents were given and if the reagent
were not given, automatically ask for saturated alcoholic picric acid
Proceed to demonstration of general tissue structure (as in question 1)
ANSWER (b)
TITLE: DEMONSTRATION OF FERROUS IRON (AND FERRIC IRON)
GIVEN: Tissue section Y
REQUIRED TO:
METHOD: (TIRMANN-SCHMELTZER)
PROCEDURE:
For Pre-treated slide
1. The section was taken down to distilled water (i.e 100%, 90%, 70% and water)
2. It was treated for 1-3hr in a dilute ammonium sulphide
For already treated slide already incubated for 3hrs in ammonium sulphide (start from step 3)
3. The section was rinsed with distilled water.

4. It was treated for 15 minute in a freshly prepared solution of equal part of 20% potassium
ferri-cyanide and 1% HCl
5. The section was rinsed with distilled water
6. Then it was Counter stained with PPB
7. Dehydrate clear and mount on DPX
RESULT
 Ferrous and ferric iron…….Dark blue
 Other pigment…….natural colour
59
 Background…….red
NOTE:
 QR is dependent upon the reduction of ferric iron to ferrous iron with ammonium
sulphide (ferric iron, [fe3+) + ammonium sulphide  ferrous sulphide [fe2+])
 The ferrous sulphide formed is converted into ferrous ferricyanide (TBB)
 QR + TBB  Tirmann-Schmeltzer
QUESTION 3
A section of paraffin embedded tissue (X) was stained to demonstrate the general tissue
structures. Examination by Histopathologist revealed traces of fine granules of formalin
pigment. You are provided with some ribbon sections of X (X 1 and X2), with the available
reagents use the most appropriate technique to:
a. stain X1 for ferric irons and

b. demonstrate the presence of ferrous salts in section X2
Answer (3a)
DATE: 27/11/2017
TITLE: DEMONSTRATION OF FERRIC IRON (Fe3+)
GIVEN: Section X1, 2% aqueous potassium ferro-cyanide, 2 % aqueous hydrochloric acid, 1 %

aqueous neutral red.
REQUIRED TO: demonstrate ferric iron in section X1
METHOD: PERL'S PRUSSIAN BLUE FOR FERRIC IRON (Using solution of potassium ferrocyanide)
PRINCIPLE: Ferric iron(fe+3) is released from any attachments to protein by treatment with
dilute hydrochloric acid, which then reacts with a dilute solution of potassium ferrocyanide to
produce an insoluble blue compound, ferric ferrocyanide (Prussian blue).
4FeCl3 + 3K4Fe (CN) 6 Fe4 [Fe (CN) 6]3 + 12KCl
PROCEDURE:
1. The section was deparaffinized and hydrated to distilled water.
60
2. Equal volumes of 2% HCl and 2% ferrocyanide solution were mixed (25.0 ml of 2%

potassium ferrocyanide + 25.0 ml of 2% hydrochloric acid) and the section was
transferred for 20 - 30minutes (report absolute value not range e.g for 20 minutes)
3. It was washed well in water.
4. It was then counterstained with neutral red lightly (2-3min) or safranin for 7minutes
5. It was washed in water.
6. The section was dehydrated rapidly in ascending grades of alcohol.
7. It was then Cleared in xylene and mounted.
RESULT:
Hemosiderin and ferric salt deposits……………blue or purple.
Cell nuclei………………….red
Background………………….pink
PRECAUTIONS
1. Avoid treatment of tissue with citrate or iron containing fluid
2. Distilled water must be iron free
3. HCl must be of analar grade
Answer (3b)
TITLE: DEMONSTRATION OF FERROUS IRON (Fe2+)
GIVEN: Section X2, 20% aqueous Potassium Ferri-cyanide, 2 % aqueous hydrochloric acid [or 1%
Glacial Acetic Acid (GAA).], 1 % aqueous neutral red.
REQUIRED TO: demonstrate ferrous iron (Fe2+) in section X2
METHOD: TURN BULLS BLUE (using Acidic solution of potassium ferricyanide)
PRINCIPLE: Tissue sections are treated with an acidic solution of potassium ferricyanide, any
ferrous iron present will react to form an insoluble bright blue pigment called Turnbull’s blue
(ferrous ferricyanide).
3FeCl2 (ferrous iron) + 2K3Fe (CN) 6 (potassium ferricyanide) Fe [Fe (CN)] 2 (ferrous
ferricyanide) + 6KCl (potassium chloride).PROCEDURE:
1. The section was deparaffinized and hydrated with distilled water.

2. The section was treated for 5-15 minutes in a freshly prepared solution of equal parts of
20% potassium ferricyanide and 2% HCL
61
3. It was then washed in 1% HCl for 5-15min (or 1% Glacial acetic acid.)
4. It was then rinsed with distilled water
5. The Section was Counterstain in nuclear-fast red for 5 minutes.
6. It was rinsed well in distilled water.
7. It was then dehydrated, cleared and cover slipped.
RESULT:
Ferrous iron…………….. Blue
Background……………….. Pink-red
QUESTION 4
You are provided with labelled samples M and N. determine the end point of decalcification
i. Report your findings ii.

Comment on your observation iii.
What method is commonly used
ANSWER (4)
TITLE: DETERMINATION OF END-POINT OF DECALCIFICATION
GIVEN: solution M and N
REQUIRED TO: determine the decalcification end point of solution labelled M and N.
METHOD: depends on the reagent provided
1. Chemical method
a. Routine/general method
b. Modified chemical method
c. Allens and Rosen
2. Physical method/manual/photometric (e.g probing tissue with nail)
3. Radiologic method
METHOD 1(a): GENERAL CHEMICAL METHOD

PRINCIPLE:
GIVEN: conc Ammonia, pipette, test tubes, test-tube rack, litmus paper, beaker, mineral acid.
62
PROCEDURE:
1. 5ml of used decalcifying fluid was added in a test tube

2. A piece of litmus paper was placed
3. conc HNO3 was added drop wisely until the medium turned alkaline
4. Turbidity or clearance was observed
OBSERVATION:
If turbidity is present, indicate the presence of calcium in excess and if clear; indicate absence
RESULT:
SAMPLE RESULT
M Clear then turbid
N turbid
METHOD 1 (b): MODIFIED CHEMICAL METHOD

PRINCIPLE:
GIVEN: Glacial acetic acid, conc. Ammonia, ammonium oxalate, pipette, test tubes, test-tube
rack, and litmus paper.
PROCEDURE:
1. 5ml of used decalcifying fluid was added in to a test tube

2. A piece of litmus paper was placed
3. Add conc NH4 drop wisely until the medium turns alkaline
4. Glacial acetic acid was added in the presence of turbidity
5. 0.5ml of ammonium oxalate was added if solution is clear
6. Turbidity was observed
RESULT:
SAMPLE RESULT
M Clear then turbid on ammonium oxalate
addition
N turbid
METHOD 1(c): ALLEN & ROSEN

PRINCIPLE:
GIVEN: ammonium oxalate, pipette, test tubes, test-tube rack, and litmus paper.
63
PROCEDURE:
1. 0.5ml of used EDTA decalcifying fluid was added in to a test tube using pipette.
2. 1.0ml of citrate phosphate (buffered to pH 3.2 – 3.6) was added
3. 2.5ml of saturated aqueous ammonium oxalate was added
4. The mixture was allowed to stand for 20minutes at room temperature 5. Turbidity or
clearance was observed
RESULT:
SAMPLE RESULT
M Clear then turbid
N turbid
SUMMARY
REAGENT TEST OBSERVATION INFERENCE CONCLUSION
Conc. NH3 strong NH3 was Solution showed solution M was Decalcification
added in drop wise alkalinity with suspected to was incomplete
2+
to solution M while red litmus paper contain Ca ions in solution M
checking for pH and become
reaction cloudy due to ppt
of CaOH at an
alkaline pH.
Conc NH3 strong NH3 was there was no solution M was There was
added in drop wise cloudiness of suspected to probably
to solution M while solution M I.e contain no Ca2+ complete
checking for pH clear ion decalcification in
reaction solution M
Saturated saturated aqueous There was solution M was There was
aqueous ammonium oxalate cloudiness of suspected to incomplete
ammonium solution was added solution M with contain plenty decalcification of
oxalate to solution M and sat. aqueous of Ca2+ bone tissue in
solution incubate at room solution of solution M
temperature for ammonium
30min oxalate
saturated saturated aqueous There was no solution M was Complete
aqueous ammonium oxalate turbidity of suspected to decalcification of
64
ammonium solution was added solution M with contain trace of bone tissue
oxalate to solution M and sat. aqueous Ca2+
solution incubate at room solution of
temperature for ammonium
30min oxalate
CONCLUSION: (Depending on your result)

The end point of complete decalcification has been achieved in M & N or the end point of
complete decalcification has not been achieved in M & N
COMMENT: (Depending on your result and method used)
For General chemical method = tissue treated in M and N should be taken to 70% alcohol and
proceeds with tissue processing. Or
For Allen & Rosen method = tissue should be taken to a freshly prepared 20% EDTA decalcifying
fluid for 2days for confirmation
QUESTION 5
Solution Q, R and T are decalcifying fluids. With the materials produced, determine the
presence or otherwise of calcium ions in the solution.
ANSWER (4).
DATE: 24/10/2017
TITLE: DETERMINATION OF END POINT OF DECALCIFCATION
REQUIRED TO: Determine the presence or absence of calcium ions in the solutions.
GIVEN: Three solutions marked Q R and T
METHOD: General chemical method
PRINCIPLE:
PROCEDURE:
TEST OBSERVATION INFERENCE
Litmus paper was dipped into Sample Q changed blue Sample Q is an acidic
sample Q,R and T litmus to red. solution. sample R is
Sample R changed red litmus an alkaline solution
to blue. sample T is an acidic solution
sample T does not change
65
from red
Ammonium solutions was sample Q shows turbidity Ca2+ present large quantity
added to the samples (Q,R in sample Q
and T) and were shaken
sample R shows no turbidity Ca2+ might not be present in
sample R and T
sample T shows no turbidity
0.5ml of ammonium oxalate Sample R shows no turbidity. Ca2+ is present in sample R
was added to samples (R and
T) and were allow to stand for Sample T shows turbidity Ca2+ trace is present in
30minutes. after the 30minutes. sample T
COMMENT:
sample Q, R and T which shows the presence of Ca2+ confirmed that the tissues were not
properly decalcified. Therefore, they should be taken back for decalcification again.
QUESTION 6
You are provided with a dense fibrous freshly removed tissue biopsy. Gross examination
revealed evidence of decalcification. With the available reagents, carryout and report all the
processes involved to ensure complete paraffin impregnation.
ANSWER (5)
DATE: 24/10/17
TITLE: TISSUE PRROCESSING
REQUIRED TO: replace the clearing agent completely with paraffin wax (impregnation media)
GIVEN: dense fibrous freshly removed tissue biopsy PROCEDURE:
PROCESS SOLUTION TIME
Dehydration 70% Alcohol 60min
Dehydration 90% Alcohol 45min
Dehydration absolute alcohol 45min
clearing xylene 60min
66

infiltration paraffin wax 30min
CONCLUSION:
The tissue is ready to proceed with embedding and sectioning.
QUESTION 7
You are provided with thin section of bone of 3-5mm. select and mount onto a suitable fixing
agent (X, Y and Z).
QUESTION 8
X is a paraffin fiber section. Carryout and report all the necessary preliminary treatments to
expose the section to demonstrate elastic fibers using alcoholic stain.
ANSWER (8)
TITLE: DEMONSTRATION OF ELASTIC FIBERS
GIVEN: Alcoholic hematoxylin, mordant (2% ferric chloride), xylene, grades of Alcohol, distilled
water, counter stain (van Gieson stain)
REQUIRED TO: Demonstrate elastic fibers in section using Alcoholic stain
METHOD: Verhoeffs van gieson stain
NOTE:
In every tissue given ask if the tissue is depigmented. If yes, you continue with the procedure,
but if not, you ask which type of chemical used for fixing the tissue, and if told you have to
report the procedure for depigmentation.
PRINCIPLE: The tissue is stained with a regressive hematoxylin, consisting of ferric chloride and
iodine. The differentiating is accomplished by using excess mordant (ferric chloride) to break
the tissue-mordant dye complex. The dye will be attracted to the larger amount of mordant in
the differentiating solution and will be removed from the tissue. The elastic tissue has the
strongest affinity of the iron- hematoxylin complex and will retain the dye longer than the other
tissue elements.
67
PROCEDURE:
1. The tissue section given was dewaxed using xylene

2. The tissue section was brought to water using alcohol in descending order.
3. Section was rinsed with distilled water
4. The tissue section was stained with alcoholic Hematoxylin for 15min
6. Section was differentiated using 2% ferric chloride for few seconds, (check
microscopically for black fibers on a gray background.)
8. Section was counterstained using van Gieson stain for 1minute
10. Section was dehydrated using alcohol in ascending order
11. Section was clear using xylene for 3 changes
12. Section was mounted using DPX or Canada balsam (whichever one given)
13. Section was examined under the microscope using 10X then 40X objective
RESULT:
 Elastic fiber………blue-black to black

 Nucleus……….blue to black
 Collagen…….red,
 Other tissue elements…….yellow e.g RBC muscle fiber etc.
QUESTION 9
Stain section marked Y and Z to demonstrate the following
Y – General tissue structure
Z - Haemosiderin
ANSWER (9)
TITLE: DEMONSTRATION OF HAEMOSIDERIN
REQUIRED TO: To demonstrate Haemosiderin in tissue section
GIVEN: Tissue slide labelled Z, 2% ferrocyanide, 2% HCL, distilled water, 1% neutral red, xylene
ascending and descending grades of alcohol DPX/Canada balsam,
68
METHOD: Perl Prussian s Blue staining

PROCEDURE:
1. The tissue section was dewaxed using xylene

2. The tissue section was hydrated using descending grade of alcohol
3. The tissue section was rinsed in distilled water
4. Equal volume of 2% ferrocyanide and 2% HCL acid were mixed and was used to stain
the tissue slide for 30 minutes
6. The tissue section was counter stained with 1% neutral red for 3minutes
8. The tissue section was dehydrated using ascending grade of alcohol
9. The tissue section was clear using xylene at 3 changes
10. The tissue section was mounted using DPX/Canada balsam
11. The tissue section was then examined under the microscope using 10X and 40X
objective
RESULT
Nuclei…….Red
Haemosiderin……..Blue
CHEMICAL PATHOLOGY
QUESTION 1
You are provided with a urine samples H, I, J and K from various patient in male medical ward
AKTH. Using the reagents provided, test for the presence of pathological sugars and protein.
Comment on your result
(if Reagents provided = BQR, clinistix strip, seliwanoffs reagent and sulphur salicylic acid)
ANSWER (1)
TITLE: URINALYSIS
AIM: To test for the presence of pathological sugars (reducing substances, fructose, glucose,
lactose) and protein in the given sample.
69
MATERIALS: Pasteur pipette, test tubes, water bath, SSA, 33% glacial acetic acid, Rotheras
mixture, Ammonium sulphate, 10% ferric chloride
PROTOCOL:
MACROSCOPY
SAMPLE COLOUR ODOUR TRANSPARENCY
H Amber pungent clear
I yellow irritating slightly turbid
J amber irritating turbid
K pale amber pungent cloudy
CHEMICAL ANALYSIS
TEST OBSERVATION INFERENCE
TEST FOR REDUCING SUBSTANCES USING BENEDICTS QUALITATIVE REAGENT
METHOD samples H,I, and J samples H, I and J

To 0.5ml of urine samples H,I,J and K in 4 turned from blue to contain reducing
different chemically clean test tubes were brick red substance
added 4.5ml BQR each
they were mixed and boiled for 10minutes sample K show no sample K contains
colour change no reducing
substance
CONFIRMATORY TEST FOR GLUCOSE USING CLINISTIX STRIP
To a fresh amount of urine samples H,I and J there was a colour glucose is
present in their respective test tubes was dipped a change from pink to in sample
H clinistix strip, removed and tapped gently at blue in sample H
the edge of the container and was compared glucose is not
with the chart within 60secs each No change in colour in present in samples
samples I and J I and J
TEST FOR FRUCTOSE USING SALIWANOFFS REAGENT
To 0.5ml of urine samples H,I and J in a samples I and J gave fructose is present
chemically clean test tubes, were added 4ml of red colour in sample H and J
seliwanoffs reagent each, mixed and boiled for
10minutes. absence of
no change in colour in fructose in sample
sample H H
COMFIRMATORY TEST FOR FRUCTOSE USING SELIWANOFFS REAGENT USING 0.2% OF
METHYLAMINE HYDROCHLORIDE & 10% NAOH
70
To 0.5ml of urine samples that showed sample changed to red Lactose present
Benedicts Qualitative test positive in a or pink colour
chemically clean test tube(s) was added 1ml of
0.2% methylamine hydrochloride and 1ml of sample did not change Lactose absent
NaOH, mixed and boiled for 10minutes colour
TEST FOR PROTEIN USING 25% SULPHUR SALICYLIC ACID (SSA)

To 5ml of urine samples in a chemically clean turbidity or white protein may be
test tubes was added to drops of 25% SSA and precipitate present
mixed
no turbidity or white protein is absent
precipitate
COMFIRMATORY TEST FOR PROTEIN USING 33% GLACIAL ACETIC ACID
To 5ml of urine sample in a chemically clean turbidity resisted protein is present
test tubes, the upper part of the urine sample
(s) was heat for 5minutes protein is absent
turbidity disappeared
add small amount of 33% GAA in a slanting
position
DETECTION OF KETONE BODIES USING ROTHERA’S REAGENT AND CONC. AMMONIUM
SULPHITE.
5ml of urine sample was saturated by a purple ring colour was ketone bodies may
Rotheras mixture. observed be present
0.5ml of conc. Ammonium sulphate was
added without mixing and left on the bench
for 15min.
COMFIRMATORY TEST FOR KETONE BODIES USING 10% FERRIC CHLORIDE
To 5ml of the urine sample in a chemically the colour disappeared ketone bodies
clean test tube(s) was added 3 drops of 10% confirmed
ferric chloride mixed and boiled for 10minutes
DETECTION OF BILE SALT USING SULPHUR POWDER
To 3ml of urine sample(s) in a chemically clean the powder sank bile salt present
test tubes was sprinkled some amount of the sulphur powder powder floats bile salt
absent
CONFIRMATORY TEST FOR PENTOSE USING BIALS REAGENT
To 0.5 ml of sample H,I and J in a chemically sample H,I and J turned presence of
clean test tubes, were added 5ml of bials to green pentose in samples
reagent each, mixed and boiled for 10minutes H,I and J
TEST FOR BILURUBIN USING FOUCHETS

REAGENT
71
To 5ml of urine sample H,I, J and K in a Sample H and I turned bilirubin is

separate chemically clean test tubes were green presence in
added small amount of barium chloride (and sample H and I
dilute sulphuric, obtained mixed and filtered sample J and K did not
The sediments of each of the 4 samples were change colour there is no
added, fouchet’s reagent each. bilirubin in sample
K and J
QUESTION 2
Specimen M and N are urine samples from 2 patients suspected to have nephritic syndrome.
Examine each specimen using the reagents provided and identify the presence of ketone
bodies and proteins with confirmation. Comment on the findings.
ANSWER 2
DATE: 23/5/2019
TITLE: URINALYSIS
AIM: To test and confirmed for the presence of protein and ketone bodies
MATERIALS: Pasteur pipette, test tubes, water bath, SSA, 33% glacial acetic acid, Rotheras
mixture, Ammonium sulphate, 10% ferric chloride.
PROTOCOL:
MACROSCOPY
Colour = Amber
Odour = aromatic
Transparency = clear
Constituency = NIL
CHEMICAL ANALYSIS
TEST FOR PROTEIN USING 25% SULPHUR
SALICYLIC ACID (SSA)
To 5ml of urine samples in a chemically turbidity or white protein may be
clean test tubes was added to drops of 25% precipitate present
SSA and mixed
no turbidity or white
72
precipitate protein is absent
COMFIRMATORY TEST FOR PROTEIN

USING 33% GLACIAL ACETIC ACID
To 5ml of urine sample in a chemically turbidity resisted protein is present
clean test tubes was a small amount of 33%
GAA in a slanting position without mixing. turbidity disappeared protein is absent
the upper part of the urine sample (s) was
heat for 5minutes
DETECTION OF KETONE BODIES USING
ROTHERA’S REAGENT AND CONC.
AMMONIUM SULPHITE.
5ml of urine sample was saturated by a purple ring colour was ketone bodies may be
Rotheras mixture. observed present
0.5ml of conc. Ammonium sulphate was
added without mixing and left on the bench
for 15min.
COMFIRMATORY TEST FOR KETONE
BODIES USING 10% FERRIC CHLORIDE
To 5ml of the urine sample in a chemically the colour disappeared ketone bodies
clean test tube(s) was added 3 drops of confirmed
10% ferric chloride mixed and boiled for
10minutes
QUESTION 3
You are provided with a chloride standard Q and a standardized mercuric nitrate solution R.
use solution R to estimate the chloride content (as NaCL) of serum samples S, T and U.
express your result in both S.I unit and traditional units. Make a comment.
Method: into tubes labelled test and standard, dispense 1.8ml of distilled water and 2ml of
standard solution respectively. Add 0.2ml of sample to the test. Add 3-4 drops of indicator to
all tubes mix and titrate to end point using solution R.
(Na = 23, Cl = 35.5 H= 1)
ANSWER (3)
DATE: 3/3/2018
TITLE: CHLORIDE ESTIMATION
AIM: to determine chloride content of given samples S, T and U
73
METHOD: Schales and schales

PRINCIPLE: When mercuric nitrate or silver nitrate solution is added to a solution containing
chloride, unionized but soluble mercuric chloride is formed. At the endpoint, the first excess
mercuric ions combine with the indicator diphenylcarbazone to give violet blue coloured
complex.
MATERIALS: Test tubes, colour reagent (diphenylcarbazone), distilled water, silver nitrate, 2ml
pipette.
PROTOCOL:
SAMPLE S SAMPLE T SAMPLE U
Titration 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
vol. of D/Water 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8
vol of sample 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8
drop of diphenylcarbazone 3 3 3 3 3 3 3 3 3
volume of AgNO3 4.00 3.00 4.00 6.00 6.00 7.00 4.00 5.00 5.00
Titre of AgNO3 3.67 6.33 4.67
RESULT:
Titre of standard = 6.0
Titre of Sample S = 3.67
Titre of sample T = 6.33
Titre of sample U = 4.67
CALCULATIONS:
X conc. Of STD.
Concentration of standard = 100 (ask for it)
Chloride concentration in sample S = 61.16 mmol/ (S.I unit)
Chloride concentration in sample T = 105.5 mmol/L
Chloride concentration in sample U = 77.8 mmol/L

Conversion to traditional unit
Since we are to estimate the chloride content (as NaCL)
Molar mass of NaCL = (23 + 35.5) = 58.5 g/mol
74
To convert to mmol/L =
Hence
Mg/dL =
Chloride concentration in sample S = 357.79mg/dL
Chloride concentration in sample T = 617.18mg/dL
Chloride concentration in sample U = 455.13mg/dL
Meq/L =
BUT equivalent weight =

Since molar mass of NaCL = 58.5 and valency is 1
Equivalent weight =
Since valency = 1
Meq/L = mmol/L
COMMENT:
The serum chloride levels of sample S, T and U Calculated above show values of 61.16mmol/L,
105.5mmol/L and 77.8mmol/L respectively. Sample S was below the normal range (Normal
range in plasma =98 – 108mmol/L.) while sample T and U are within the normal range. Hence
the patient has a normal value of sodium chloride.
QUESTION 4
Estimate the chloride content of sample Q provided using the method
I. Pipette 1.8ml of distilled water in a given test tubes marked “Test” and “Standard” II.
Add 0.2ml of sample and 2ml of standard solution into respective containers.
III. Add 3 drops of indicator solution to end point
IV. titrate the samples five times and find
a. mean
b. S.D
75
c. CV
Comment on your result
ANSWER (4)
DATE: 23/10/2017
TITLE: CHLORIDE ESTIMATION
AIM: to determine chloride content of given sample Q
METHOD: Schales and schales
PRINCIPLE:
MATERIALS: Test tubes, 2ml pipette, silver nitrate, colour reagent (diphenyl carbazone),
distilled water
PROTOCOL:
No of test tubes T1 T2 T3 T4 T5 STD
Vol. of D/water (ml) 1.8 1.8 1.8 1.8 1.8 1.8
vol. of sample S (ml) 0.2 0.2 0.2 0.2 0.2 0.2
vol. of STD (ml) - - - - - 0.2
drops of indicator 4 4 4 4 4 4
volume of AgNO3 0.28 0.29 0.28 0.29 0.28 1.37
It was mixed and titrated to end point using Ag (NO3)2
RESULT:
Titre of standard = 1.37
TITRATION Result
first 0.28
second 0.29
third 0.28
fourth 0.28
fifth 0.29
CALCULATION
76
x conc. Of STD.
Chloride conc in T1 mmol/L
Chloride conc in T5 21.20 mmol/L

ANSWER (4a)
The mean (x) of the titre (x) = = 20.7 = conc. Of chloride in sample S
To calculate the mean deviation
x x-x(bar) (x-x)2
20.4 20.4 – 20.7 = -0.3 0.09
21.2 21.2 – 20.7 = 0.5 0.25
20.4 20.4 – 20.7 = -0.3 0.09
20.4 20.4 – 20.7 = -0.3 0.09
21.20 21.2 – 20.7 = 0.5 0.25
∑ (x-x)2 = 0.77
n=5
ANSWER (4b)
ANSWER (4c)
C.V = = 2.12%
Precision =
NOTE:
 Coefficient of variation above 5% give low or poor precision

 The lower the coefficient of variation the higher the precision
77
COMMENT: Based on the concentration obtained and the statistical analysis, there is high
precision of method used. The method of chloride estimation shows a precision of 0.47 Normal
range in plasma =98 – 108mmol/L.
QUESTION 5
Determine the bicarbonate content of serum samples S. report your findings and make a
comment.
Method: Pipette 2.0ml of distilled water in a conical flask. Add 1ml of 0.01NHCL. Add 0.2ml of
plasma/serum and shake well to liberate CO2. Add 1-2 drops of neutral red. Titrate the
mixture against 0.01N NaOH
ANSWER (5)
TITLE: BICARBONATE ESTIMATION
AIM: To determine serum bicarbonate level in the given sample
MATERIALS: 0.01N HCL acid and 0.01N NaOH, distilled water, serum, test tubes, neutral red
indicator,
METHOD: Van Slyke method
PRINCIPLE: when 1ml of 0.01N HCL is mixed with serum sample (0.2ml), part of the acid will be
neutralized by the serum. The excess acid is titrated against 0.01N NaOH. The resultant volume
remaining is proportional to the amount of bicarbonate neutralized by HCL
2HCL + HCO3  H2O + CO2 + HCL (excess)
HCL (excess) + NaOH  H2O +NaCL
PROTOCOL:
TEST STANDARD
Distilled water (ml) 2.0 -
HCL (ml) 1.0 1.0
Serum (ml) 0.2 -
Neutral red (drop) 1 1.0
CALCULATION:
Volume of HCL utilized by HCO3 = (1-y) ml
Conc. = (1-y) x 50 where y = titre
78
RESULT
Titre 1 = y1 = 0.36ml
Titre 2 = y2 = 0.56ml
Conc = (1-y) x 50
1st conc = (1-0.36) x 50 = 32mmol/L
2nd conc = (1-0.56) x 50 = 22mmol/L

COMMENT: (Normal range = 24-33mmol/L)
The serum bicarbonate levels calculated above show values of 32 and 22 mmol/L. the first
sample is within the normal range while the second sample shows decreased level of serum
bicarbonate.
QUESTION 6
Determine the bicarbonate content of sample Q provided using the method:
Pipette 2.0ml of distilled water in a conical flask. Add 1ml of 0.01NHCL. Add 0.2ml of
plasma/serum and shake well to liberate CO2. Add 1-2 drops of neutral red. Titrate the
mixture against 0.01N NaOH (1ml) five times
a. mean
b. Standard deviation (S.D)
c. Coefficient of variation (CV)
Comment on your result with regards to precision of the method.
ANSWER 6
TITLE: BICARBONATE ESTIMATION
AIM: To determine serum bicarbonate level in the given sample
MATERIALS: 0.01N HCL acid and 0.01N NaOH, distilled water, serum, test tubes, neutral red
indicator,
79
METHOD: Van Slyke method

PRINCIPLE: when 1ml of 0.01N HCL is mixed with serum sample (0.2ml), part of the acid will be
neutralized by the serum. The excess acid is titrated against 0.01N NaOH. The resultant volume
remaining is proportional to the amount of bicarbonate neutralized by HCL
2HCL + HCO3  H2O + CO2 + HCL (excess)
HCL (excess) + NaOH  H2O +NaCL
PROTOCOL:
T1 T2 T3 T4 T5 STD
Distilled water (ml) 2.0 2.0 2.0 2.0 2.0 -
HCL (ml) 1.0 1.0 1.0 1.0 1.0 1.0
Serum (ml) 0.2 0.2 0.2 0.2 0.2 0.2
Neutral red (drop) 1 1 1 1 1 1
CALCULATIONS:
Volume of HCL utilized by HCO3 = (1-y) ml
RESULT:
Titre of standard = 0.75ml
TEST 1st titre 2nd titre average titre
sample A (ml) 0.60 0.62 0.61
sample B (ml) 0.53 0.49 0.51
sample C (ml) 0.59 0.61 0.60
sample D (ml) 0.47 0.47 0.47
sample E (ml) 0.50 0.52 0.51
Concentration of samples
Conc of sample A = (1-0.61) x 50 = 19.5mmol/L
Conc of sample B = (1-0.51) x 50 = 24.5mmol/L
Conc of sample C = (1-0.60) x 50 = 20.0mmol/L
Conc of sample D = (1-0.47) x 50 = 26.5mmol/L
80
Conc of sample E = (1-0.51) x 50 = 24.5mmol/L
ANSWER 6(a)
Mean conc (x) = 23.0

To calculate the mean deviation
x x-x(bar)
19.5 19.5 – 23.0 = -3.5
24.5 24.5 – 23.0 = +1.5
20.0 20.0 – 23.0 = -3.0
26.5 26.5 – 23.0 = +3.5
24.5 24.5 – 23.0 = +1.5
n=5
ANSWER (6b)
ANSWER (6c)
C.V = = 12%
Precision =
COMMENT: (Normal range = 24-33mmol/L)
Based on the concentration obtained and the statistical analysis, there is high precision of
method used. The method of bicarbonate estimation shows a precision of 0.08 which is below 1
(precised)
QUESTION 7
Estimate the urea content of the serum sample K using procedure below. Urea standard is
50mg%.
Method: dilute sample K and standard given 1:100 with distilled water. Dispense 1ml of
distilled water into tubes (test and standard) and 2ml of distilled water in to blank tube. Add
1ml of the diluted sample and standard into their corresponding tubes. Add 1ml each of
81
colour and acid reagents to all tubes and mix thoroughly and heat immediately in a hot water
bath for 15mins. Allow to cool for 5mins and read the absorbance at 520nm against blank.
ANSWER 7
TITLE: UREA ESTIMATION
AIM: To determine the concentration of urea in the given sample
MATERIALS: test sample, distilled water, test tubes, test tube rack, urea colour, urea acid,
Pasteur pipette, micropipette, and spectrophotometer.
METHOD: Diacetyl monoxime method (DAM)
PRINCIPLE: When urea is heated in strongly acidic conditions with substance such as
diacetylmonoxime, yellow to pink colour complex is formed is formed which is read at 520nm
using a spectrophotometer.
Urea + Diacetyl monoxime (DAM) Diacetyl-Urea
Diacetyl-Urea + Fe3+ +acidic pH Yellow Diazine (measured at 520 )

PROTOCOL:
STAGE 1: A 1 in 100 dilution was made
TEST STANDARD
Sample (ml) 0.02 -
standard (ml) - 0.02
distilled water (ml) 1.98 1.98
STAGE 2:
TEST (K) STD BLANK
Diluted sample (ml) 1.0 - -
dilute standard (ml) - 1.0 -
distilled water (ml) 1.0 1.0 2.0
urea colour (ml) 1.0 1.0 1.0
urea (acid) (ml) 1.0 1.0 1.0
It was mixed thoroughly and incubated in a water bath for 20minutes. It was allowed to cool for
5minutes and was read at 520nm spectrophotometrically
RESULT:
82
[STD] = 50mg% (10 mg/L urea BUN divided by 0.466 = 21 mg/L urea = 0.36 mmol/L
urea.)
But [STD] mmol/L = = 8.33mmol/L or mg/dL x 0.1665 = mmol/L

TEST (K) STANDARD BLANK
absorbance 0.056 0.157 0.000
𝐴𝑏𝑠 𝑜𝑓 𝑡𝑒𝑠𝑡 0.056
[Urea] = 𝑥 [𝑠𝑡𝑑] = 𝑥 8.33 = 2.97mmol/L
𝐴𝑏𝑠 𝑜𝑓 𝑠𝑡𝑑 0.157
CONCLUSION: Sample K was found to be 2.97mmol/L which is within the normal range
Normal range:
Serum: 15- 45 mg/dL (2.49-7.49 mmol/L),
Urine = 20 - 35 gr/24 h,
Blood Urea Nitrogen (BUN) = 8 – 25 mg/dl
Urea is the final result of the metabolism of proteins; it is formed in the liver from its
destruction. Elevated urea can appear in blood (uremia) in: diets with excess of proteins, renal
diseases, heart failure, gastrointestinal hemorrhage, dehydration or renal obstruction. Clinical
diagnosis should not be made on a single test result; it should integrate clinical and other
laboratory data.
QUESTION 8
Estimate the Creatinine content of sample Q provided using the given method:
ANSWER (8)
DATE: 27/11/2017
TITLE: CRETININE ESTIMATION
AIM: To determine the concentration of creatinine in the given sample.
METHOD: Jaffes Method
83
PRINCIPLE: Creatinine reacts with picric acid in alkaline medium to produce orange to red
colour complex which is read spectrophotometrically at 490- 500nm
Creatinine + Picric acid + alkaline pH 2,4,6 trinitrophenol (Janovski’s complex)
PROTOCOL:
STAGE 1: Deproteination stage
TEST
H2O (ml) 1.0
Serum (ml) 0.25
10% sodium tungstate (ml) 0.25
2/3 NH2SO4 (ml) 0.5
It was mixed and allowed to stand for 5minutes.
STAGE 2:
TEST ( ) STANDARD BLANK
Filtrate (ml) 1.0 - -
distilled water 1.0 1.75 2.0
standard (ml) - 0.25 -
picric acid (ml) 0.5 0.5 0.5
0.75 NaOH (ml) 0.5 0.5 0.5
It was mixed and allowed to stand at room temperature for 15minutes and then read
spectrophotometrically at 500nm RESULT:
TEST BLANK STD
0.14 0.14 0.00
CALCULATION:
[STD] = 4mg%
[STD] mmol/L = = 0.40mmol/L
[Creatinine] = x 0.40 = 0.45mmol/L

CONCLUSION: Serum creatinine estimation was conducted and the result obtained was above
the normal range
Normal range
Male = 0.7 - 1.3mg/dL or 0.06-0.125mmol/L or (61.8 – 123.7) µmol/L
84
Female: = 0.6 – 1.1mg/dL 0r 0.05-0.101mmol/L or (53.0 – 97.2) µmol/L

CLINICAL SIGNIFICANCE
Creatinine is the result of the degradation of the creatine, component of muscles, it can be
transformed into ATP that is a source of high energy for the cells. The creatinine production
depends on the modification of the muscular mass, and it varies little and the levels usually are
very stable. Is excreted by the kidneys. With progressive renal insufficiency there is retention in
blood of urea, creatinine and uric acid. Elevate creatinine level may be indicative of renal
insufficiency. Clinical diagnosis should not be made on a single test result; it should integrate
clinical and other laboratory data.
QUESTION 9
Determine the random blood sugar content of the specimen’s marked S and U using method
provided.
ANSWER (9)
TITLE: BLOOD GLUCOSE ESTIMATION
AIM: To determine the random blood sugar content of given samples/ specimens S and U.
MATERIALS: test tubes, test serum, glucose reagent, standard or control, distilled water,
Pasteur pipette, micropipette, test tube rack, spectrophotometer.
METHOD: Glucose oxidase method
PRINCIPLE: Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid and
hydrogen peroxide. In the presence of enzyme peroxidase, released hydrogen peroxide is
coupled with phenol and 4-Aminoantipyrine (4-AAP) to form coloured Quinoneimine dye.
-D-glucose +H2O+O2 Glucose oxidase D-gluconic acid+H2O2

H2O2+ phenol + 4AAP Peroxidase
Quinonemine dye +4 H2O +
The intensity of the color formed is proportional to the glucose concentration in the
sample.
PROTOCOL:
No of test tubes Test (S) Test (U) Standard Blank
glucose colour reagent 1.0 1.0 1.0 1.o
(ml)
serum/plasma (ml) 0.01 0.01 - -
standard solution (ml) - - 0.01 -
Distilled water (ml) - - - 0.01
85
It was mixed and incubated at 37oC for 5minutes. The absorbance of test and standard was read
against the blank spectrophotometrically at 510nm.
RESULT:
No of test tubes Test (S) Test (U) Standard Blank
optical density (OD) 0.09 1.36 0.13 0.00
CALCULATIONS:
X conc of STD (where conc of STD = 5.6mmol/L)
But = 5.56mmol/L or mg/dL x 0.0555= mmol/L (MW of Glucose =180)
For sample S x 5.56

= 3.85mmol/L
For sample U x 5.56 =

58.16mmol/L
Concentration of blood sugar in sample S is 3.85mmol/L Concentration
of blood sugar in sample U is 58.16mmol/L
COMMENT:
The normal range for:
Random blood sugar is 3.9 – 7.0 mmol/L
FBS = 3.9 – 8.0 mmol/L
Patient S has a normal blood sugar concentration
Patient U is hyperglycemic
CLINICAL SIGNIFICANCE
Glucose is a major source of energy for most cells of the body; insulin facilitates glucose entry
into the cells. Diabetes is a disease manifested by hyperglycemia; patients with diabetes
demonstrate an inability to produce insulin. Clinical diagnosis should not be made on a single
test result; it should integrate clinical and other laboratory data.
86

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First Professional Questions and Answers by Hon. Ukpor Iyang

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HON.

UKPOR IYANG IYANG (NIMELSSA PIKIN)

FIRST PROFESSIONAL QUESTIONS AND ANSWERS

AIM: To identify the malaria parasite in sample marked “A”

1. I ensured accurate and proper timing during staining 2.

 No ova, cyst or vegetative form of parasite seen

AIM: To identify intestinal parasites present in the sample labelled F.

Parasites found in faeces

Parasites found in urine and blood

TITLE: THIN FILM MAKING

1. Sample N was mixed properly

 Neutrophils, eosinophils………….pale pink

 Malaria pigment (in monocytes)………brown – black

 Macrocyte i.e when cell diameter > 8µm

1. A thin film was made

Granules of eosinophils . . . . . . . . . . . . . . . . . . . Red

1. A thick film was made on a slide.

a. Describe the appearance of the bacterial colony.

1. The wireloop was sterilized to red hot and allowed to cool.

TITLE: MOTILITY TEST

Call the supervisor for initials.

1. A quantity of the urine was aseptically transferred into a centrifuge tube.

 Pus cells……………….…… seen

 I avoided over decolorization with acetone

a. Carryout a gram staining technique on different, colonies present on the plate.

S/N CHARACTERISTICS COLONY 1 COLONY 2 COLONY 3

1. 2-4ml of hydrogen peroxide was poured into a test tube.

TITLE: COAGULASE TEST

 Clumping of the suspension seen after few seconds = coagulase positive

ON BLOOD AGAR: These are the characteristics of this bacteria

shape round round round irregular round round

Parameters S. aureus E.coli Kleb proteus pseudo strep

COMMENT: it was observed that sample C contains gram Positive bacteria

TITLE: CATALASE TEST TITLE: OXIDASE TEST

TITLE: COAGULASE TEST TITLE: MOTILITY TEST

CONCLUSION: true motility was seen which

TITLE: INDOLE TEST

a. Prepare a nutrient agar plate containing 1/50,000 dilution of rystal violet

R = 1/50,000 V= 20ml O = 1/100

1. The agar was melted in a hot water bath

R = 10% V = 20ml O = 100%

1. 2ml of the molten media was withdrawn and discarded aseptically

1. 1.004ml of molten media provided was withdrawn aseptically and discarded

3. It was pour into a clean sterile labelled petri dish

R = 2% of blood provided V = 20ml O = 100%

TITLE: SPECIMEN SUBCULTURE

See gram staining above

1. A Smear was made and heat fixed

2 The plate labelled A is an overnight culture of an aspirate.

HEMATOLOGY & BGS

(+) = agglutination (-) = no agglutination

 It is a screening test for blood donation

positive control Positive(+)

(+) = agglutination (-) = no agglutination

 It is a screening test for blood donation

TITLE: INDIRECT COOMBS TEST

1. Three (3) drops of serum were added on a test tube

ii. Prothrombin ratio

iii. Prothrombin index

Prothrombin index (control to test) =

For sample L = iv.

NORMAL RANGE = 11-18sec

Vol of test tubes 1 2 3 4 5 6 7 8 9 10 11

1. 2-3 drops of the stain solution was placed in a test tube.