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Internship Report
Bachelor in Microbiology
Submitted By
Department of Microbiolgy
Pakistan, 2019
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Date _____________
FINAL APPROVAL
It is certified that we read the the enternship report by Mr Muhammad Attique (Reg No 15-F-
AWKUM-GCM-BS-MBIO-07) and Mr Adnan Ali Shah (Reg No 15-F-AWKUM-GCM-BS-
MBIO-30) and it is our judgment that this project is of sufficient standard to warrant its
acceptance by Abdul Wali Khan University Mardan for the award of BS (Hons) Degree in
Microbiology.
Supervisor
Mr. Tahir Hussain
Lecturer
Department of Microbiology
Abdul Wali Khan University Mardan ____________________
External Examiner
Mr. Fazal jalil
Lecturer
Department of Biotechnology
Abdul Wali Khan University Mardan ____________________
Dedicated
To our
Loving Parents:
The soul of our goodness
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TABLE OF CONTENTS
Acknowledgments……………………….……………………………………………………………………………………………………07
Introduction……………………………………………………………………………………………………………………………………..08
History of MMC…………………………………………………………………………………………………………………………………09
Phlebotomy………………………………………………………………………………………………………………………………………10
Microbiology…………………………………………………………………………………………………………………………………….11
Common media in routine use............................................................................................................17
18
Gram staining……………………………………………………………………………………………………………………………………19
Zn staining…………………………………………………………………………………………………………………………………………22
Hematology………………………………………………………………………………………………………………………………………24
CBC……………………………………………………………………………………………………………………………………………………25
ESR………………………….………………………………………………………………………………………….…………………………….26
Sickling test…………………………………………………………….…………………………………………………………………………27
Serology……………………………………………………………………………………………………………………………………………29
Blood grouping………………………………………………………………………………………………………………………………….30
Cross match………………………………………………………............…………………………………….…………………………….31
HBsAG……………………………………………………………………………………………………………………………………………….33
Dengue……………………………………………………………………………………………………………………………………………..34
HCV…………………………………………………………………………………………………………………………………………………..35
H pylori………………………………………………………………………………………………………………….………………………….36
Brucella…………………………………………………………………………………………………………....................................37
Pregnancy test………………………………………………………………………………………………………………………………….38
Chemical pathology…………………………………………………………………………………………………………………………..40
Complete urine analysis………………………………………………………………………………………………………….…….….41
Glucose test……………………………………………………………………………………………………………………………….…….51
Uric acid………………………………………………………………………………..…………………………………………………….……52
ALT…………………………………………….……………………………………………………………………………………………………..52
AST …………………………………………………………………………………………………………………………………………………..54
Molecular pathology……………………………………………………………………………………………………..………….………55
Extraction of DNA by chelex method…………………………………………………………………………………………..….…56
PCR………………………………………………………………………………………………………………….…………….……..………….56
Agarose gel electrophoresis……………………………………………………………………………………………………………...57
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LIST OF FIGURE
Figure 1: Phlebotomy............................................................................................................................13
Figure 10 sterilization.....................................................................................................................22
Figure 20 Urine sample collection sample for physical and other tests….....................................46
ABBREVATIONS
ZN Zeil Neilson
HB Haemoglobin
HCT Haematocrit
ACKNOWLEDGMENTS
All glories be to ALLAH, the most merciful and benevolent, who created the Universe
and everything in it and gave man the ability to think and search. Who ordered man to search and
learned. Our humblest and deepest obligations are due with great honor and esteem to the Holy
Prophet Hazrat Muhammad (SAW), who is a torch of guidance and knowledge for humanity as a
whole.
What can we do to just express our pleasant thanks and appreciation to our sweet teacher
and supervisor Mr. Tahir Hussain, Lecturer Department of Microbiology, Abdul Wali Khan
University Mardan, for his supervision, advice and guidance from the very early stage of this
internship as well as giving us extraordinary experiences throughout the work above all and the
most needed, he provided us unflinching encouragement and support in various ways. He
constant oasis of ideas and passions in science, which exceptionally inspire and enrich us growth
as a student, a researcher and a scientist want to be. We indebted to her more than she knows.
We are grateful in every possible way and hope to keep up our collaboration in the future.
We are very much thankful to all the staff members of the, Department of Microbiology, Abdul
Wali Khan University Mardan, especially to Dr. Muhsin Jamal, Dr. Hazir Rahman, Ms.
Kanwal Mazhar, Dr. Ziaur Rahman, Mr. Sagheer Ahmad and Ms. Aliya Khalid for their
kindness and moral support during our study.
We are wish to express our gratitude to all the staff members of MMC, for research facilitation,
clinical experience and systematic approach.
We are very much thankful to Zakir Ullah, Mansoor Ahmad, Haseeb Khan, Basit Ali khan and
Muhammad Junaid, Thanks for the friendship and memories. Last but not least, our deepest
gratitude goes to our beloved Fathers and especially to our Mothers also to our Uncles and sisters
for their endless love, support and countless prayers for our success throughout the course of life.
Thank you very much.
INTRODUCTION
Internship is an integral part of the program in laboratory medicine and to provide students with
an opportunity to integrate and apply acquired knowledge and skills in actual clinical settings.
With the help and guidance of laboratory professionals and other qualified laboratory personnel
and health professionals, students learn more about diagnostic test procedures, quality control
and instrumentation in the clinical laboratory. They also gain an understanding of the roles and
functions of the medical laboratory professionals.
The internship provides applied learning experiences during which the student should:
DEPARTMENTS OF MMC:
1. Chemical pathology
2. Haematology
3. Histopathology
4. Immunology
5. Microbiology
6. Virology
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PHLEBOTOMY
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1.PHLEBOTOMY
The collection of the blood sample is called phlebotomy and the person who collects the blood
from the patient by technique is called phlebotomist. In MMC sample collection was done in
reception. Phlebotomists collected the blood specimens from patients. Specimens were taken by
phlebotomist in central accessioning area where they were processed, logged into the computer
and given a specimen number and then distributed to the departments for the testing.
Figure 1: Phlebotomy
TYPES OF SAMPLES
1. Aseptic fluid
2. Amniotic fluid
3. Serum
4. Plasma
5. Urine
6. CSF
PROCEDURE:
1. Checked for signed, dated approved form and signed physician’s orders.
2. As certain the patient’s ID, name, date of birth.
3. Explain procedure to patient.
4. Used hand sanitizer to free hands from any type of contamination.
5. Preparation of study procedure equipment is done.
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C. INITIATION OF SAMPLING:
1. Assess the patient’s veins by palpation
a) Applied tourniquet
b) Tie tourniquet in such a way to allow release with one hand.
c) Clean skin with alcohol swab using aseptic technique.
2. Ask patient to form a fist so the veins are more prominent.
3. Enter the vein at an angle of 30 degree.
4. As blood has been collected, release the tourniquet and withdraw the needle.
5. Apply clean gauze or dry cotton to the site.
MICROBIOLOGY
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III. Chocolate Agar :Blood agar is heated for its preparation.On Chocolate agar we
culture meningococcus, pneumococcus, and Haemophilus.
VI. Blood Agar: Is used most commonly. Bacteria when grown in blood agar produce
hemolysis along their growing colonies. Some bacteria produce no hemolysis. Types
of changes:
a) Beta haemolysis, There is clear no growth zone around bacterial colony of
complete hemolysis, e.g. Streptococcus pyogenes is a beta hemolytic Streptococci.
b) Alpha haemolysis, When there is greenish discoloration around bacterial colony
colony e.g. Viridans streptococci.
c) Gamma haemolysis, or, No hemolysis. There is no change in the medium near
the bacterial colony.
The agar prepared has the same composition. The final pH of both media is 7.4.
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AUTOCLAVING
Autoclaving is a process that use pressure and heat heat and pressure to sterilize so all parts of
the material at 121 degree Celsius for 15 minutes.
Objective:
Preparation of sterile nutrient agar for culturing.
PROCEDURE
1. Required amount of broth (with agar) powder is weighed into Scott bottles and
suspended in distilled water.
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2. The bottles are loosely recap and place in autoclave for sterilization.
3. Figure 8 sterilization
4. Media is sterilized for 15 min, at 121 degree celcius.
5. Media is removed after autoclaving. Prepared broth is allowed to cool and the bottles
tightly.
In gram negative bacteria, cell wall takes up the CV-Iodine complex but due to the thin layer of
peptidoglycan, CV-Iodine complex gets washed off. Decolorizer dissolves the lipids in the cell
walls, and wash out crystal violet-iodine complex of the cells.So, when again stained with
safranin, they take the stain and appears red in colour.
SMEAR PREPARATION
1. Take a dry slide.
2. Sterilize the wire loop on Bunsen burner.
3. Transfer culture by wire loop and make a smear.
4. Air dry the smear.
5. Dry smear through flame.
PROCEDURE
1. Cover the smear with C-V stain for 1 minute.
2. Wash it by water.
3. Apply iodine solution and leave it for 1 minute.
4. Again wash the slide by water.
5. Apply decolourizer then wait for 30 seconds
6. Again wash the slide by water.
7. Apply safranin and wait for 1 minute.
8. Again wash slide by tap water.
9. Observe slide under microscope (oil immersion 100x) using a Bright field microscope.
RESULT
The staining results of gram stain are as follows:
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Figure 12 Zn staining
REAGENTS:
• Basic dye Carbol fuschin
• Malachite green
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PROCEDURE
1. Spread the sputum over the slide.
2. Air dry slide for about 30 minutes.
3. Heat fix the smear.
4. Cover the smear with carbol fuchsin stain.
5. Wash off the stain with clean water.
6. Cover the smear with 3% v/v acid alcohol for 2-5 minutes (or 20% sulfuric acid) or until
the smear is sufficiently decolorized.
7. Wash out it under tap water.
8. Apply malachite green stain over slide and wait for 1-2 minutes
9. Again wash stain with water.
10. Examine the smear under microscope and scan the smear systematically.
HEMATOLOGY
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Complete blood count is also called complete picture. In the lab of MMC CP was performed on
the hematology analyzer. The hematology analyzer was an instrument which was count all the
cell was present in the blood. It was also count the differential leukocytes count. It counts all the
cells in blood within one minute with Reference ranges and results were displayed on the
computer system.
Hb Male: 13 - 17 g/dl
Female: 12 - 15 g/dl
HCT Male: 42 - 50 %
Female: 37 - 57 %
MCV 77 - 97 Fl
MCH 26 - 33 pg
MCHC 31 - 36 g/dl
Neutrophils 39 - 74 %
Lymphocytes 20 - 44 %
Eosinophils 1-4%
Monocytes 2-6%
Basophils 0-1%
Principle:
Anticoagulated blood is left undisturbed in verticle glass tube for sometime, to allow settling of
the RBCs from the plasma. The settling rate is measured. This mechanism involves three stages:
I. Aggregation: This is first phase in which piling of red blood cell takes place. It takes 10-
15 minutes.
II. Sedimentation: This is second phase in which actual falling of red blood cells takes
place.It takes 30-40 minutes of 1 hour.
III. Packing: This is the last stage. In this, there is a slower rate of falling of sedimented
RBCs in column occurs due to overcrowding. It take place in final 10 minutes in 1 hour.
WESTERGREN METHOD
The Westregren tube has two open ends. Its length is 30cm and diameter is 2.5mm. It contains about 2 ml
of blood.
REQUIREMENTS
• Anticoagulated blood
• Westergren tube
• Westergren stand
• Rubber bulb (sucker)
PROCEDURE
1. Mix anticoagulated blood.
2. Draw the blood into the tube up to 0 mark.
3. Wipe out blood from bottom of tube with cotton.
4. Set the tube upright in stand.
5. Left the tube undisturbed for about a hour.
6. Read the result at end of 1 hour.
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NORMAL VALUE
• For males : 0-11 mm/hr
• For females : 0-14 mm/hr
PRINCIPLE:
Sodium meta-bi-sulphate reduces the oxygen tension inducing the typical sickle cell.
REQUIREMENTS:
• Cover slip
• Wax
• Microscope
• Glass slides
• Sodium meta-bi-sulphate
• Distilled water
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1. Finger is punctured with the help of needle and fresh blood sample is collected.
2. The blood samples collected is stored at 1° to 10° C and can be used for testing for up to
45 days.
Procedure:
SEROLOGY
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• Tube method
• Tile method
• Slide method
Procedure:
INTERPRETATION OF TEST:
• Negative result: One purple line appearance.
• Positive result: Two line appearance.
• Invalid result: If no purple colour line appear result is considered invalid
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Procedure:
1. Put two drops (50 µl) of plasma into the sample well ‘S’ .
2. Reas result at the end of fifteen minutes.
Interpretation:
1. If G and M line were absent and C line is formed thwn result is considered as negative.
2. If M line appeared it shows the prescence of antibody against dengue so result is
considered as positive.
3. If G line appeared it shows prescence of IgG so test is considered as positive.
4. If C band appeared but no other bands appear then result is considered Invalid.
• Stopwatch
• Patient Serum
• HCV Device
PROCEDURE:
1. Put 1 drop (10µl) of plasma to the sample well of the test card (marked “S”).
2. Put two drops of Sample Diluent to the well (marked “D”.
3. After 15 minutes note down test result.
TEST INTERPRETATION:
• Negative result: Only one purple line appeared.
• Positive result: When two colour bands appear.
• Invalid result: When no coloured line formed in the test region then result is considered
as invalid.
REQUIREMENTS
• Diluent
• Pipette
• Patient serum or whole blood
• H.pyori ICT device
PROCEDURE
1. Add 20µl of whole blood, 10µl of serum into sample well (S) on the test device and then
add assay diluents about 3 drops.
2. A purple coior is formed and moves towards the centre as the test proceed.
3. Note down result after 10 minutes.
INTERPRETATION OF TEST
PROCEDURE:
1. Take blood and centrifuge it.
2. Now take the 10ul serum and add one drop of Brucella Abortus and Brucella Melitensis
reagent in each 10ul of serum drop and mix them for about 3 min.
3. Now examine the sample and see the aggregation of cell, which show the Brucella
presence.
PREGNANCY TEST
Pregnancy test is is performed to check whether a women is pregnant or not.
PRINCIPLE:
An anti-HCG colloidal partical is suspended in membrane in the test kit. When the HCG
is present in sample passes through it leads to formation of pink colored line in test
region which shows test positivity.
SPECIMEN REQUIREMENTS:
VOLUME REQUIRED:
We prefor early morning samples because they are more concentrated and helps is providing
quality result.
STORAGE:
The sample collected should be stored in refrigerator at about 1-7°C and any additives
are avoided. Before performing test sample is brought to room temperature
PROCEDURE:
1. Check that both the sample and kit are at room temperature if have been previously
refrigerated.
2. Check the urine sample is fully labeled with patient identification.
3. Remove device from the sealed pouch.
4. Using the transfer pipette supplied with the pouch, transfer 3 full drops of urine (100ul) to
the sample well (S).
5. Read test result at 3 minutes after applying the urine sample, not before
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INTERPRETATION:
• Positive The formation or appearance of two colored lines one in control region
and the other in the test region.
• Negative Formation of line only in control region
• Invalid No color line appear.
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CHEMICAL PATHOLOGY
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Figure 19 Urine sample collection sample for physical and other tests.
CHEMICAL EXAMINATION:
Chemical examination of urine is mostly performed on redy made test strip. The most frequently
performed chemical tests using reagent test strips are:
• Specific Gravity (SG)
• pH
• Protein
• Glucose
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• Leukocyte Esterase
• Urobilinogen
• Nitrite
• Bilirubin
• Ketones
• Blood and Myoglobin
PH of urine:
Protein:
We can find out prescence of albumin in urine by protein test.Normally urine is free of protein.
• The protein was detected by the strip.
• By the changing of colour of strip the cloudiness was show the protein (albumin).
Table 3 interpretation of protein in urine
Cloudiness Result
No cloudiness Negative
Slight cloudy +
Moderate cloudy ++
BILE PIGMENTS:
BILE SALTS:
KETONES BODIES:
Normal urine has no ketone bodies. Ketones are not normally found in the urine. They are
intermediate products of fat metabolism. It is produced or released in urine due to fasting or body
inability of body to utilize carbohydrates properly.
Blood in urine:
Blood is normally absent in urine .We detect haemoglobin in urine in these test.Blood in urine
was detected by strip colour changing.
colour produced
Result
Yellow Negative
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Green ++
Microscopic examination
For microscopic examination we use urine sediments. We examine following cells and substance
in urine microscopically
• Red Blood Cells (RBCs)
• White Blood Cells (WBCs)
• Epithelial Cells
• Bacteria, Parasite and Yeast
• Crystals
RBC’s:
A few RBC’s are normally present in urine.Increase in RBC’s number in urine can be observed
under microscope. which was seen as
• Small disc shaped, yellow in color and darker at the edge about 8µm in diameter.
Urine of a healthy person collected is sterile and free from microbes the prescence of any type of
microbe is indication of infection.
SAMPLE MATERIAL:
Serum heparinized or EDTA plasma.
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PROCEDURE:
PRINCIPLE:
Uric acid is oxidize to uricase to allantoin with the formation of hydrogen peroxide. In the
presence of peroxidase (POD) a mixture of dichlorophenol sulphate (DCPS) and
4aminoantipyrine 4-(AA) is oxidized by hydrogen peroxide to form a quioneimine dye
proportional to the concentration of uric acid in the sample.
SAMPLE MATERIAL:
hemolysis free serum, EDTA or heparinized plasma and urine.
PROCEDURE:
• 1000µl of monreagent is taken in a glass tube and 10µl of plasma is added.
• Incubate for 5 min at 37c.
• Sample tube is then loaded on the analyzer after setting the required program for uric
acid.
• Results are displayed on screen.
SAMPLE MATERIAL:
Non-haemolysed serum and EDTA plasma.
PROCEDURE:
• Take 500ul reagent from reagent well, Place the test tube for 2 min at 37C.
• Add 50ul serum taken from centrifugation into reagent tube and mixed and sip the
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Procedure:
Adjust spectrophotometer to 340 nm and 25°C. Add 2.9 ml of the reagent mixture into cuvette
and place under spectrophotometer. Incubate it for 4 - 5 minutes to obtain temperature
equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted
enzyme and record the decrease in A 340 for 4 - 5 minutes. Calculate ΔA 340 /minute from the
initial linear portion of the curve.
NORMAL RANGE:
MOLECULAR BIOLOGY
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REQUIREMENTS:
• Chelex reagent
• Ependorf tubes
• Centrifuge
• Vortex
• heating block
PROCEDURE
• Make 6-7% solution of chelex reagent.
• Aliquot 300uL solution in 15ml ependorf tube.
• Take 300uL blood and 3mL distilled water or RBC lyses solution.
• Subject it to centrifuge at 5000 rpm for 2 minutes.
• Repeat lyses step.
• Add 300uL 5-7% chelex solution.
• Subject it to Vortex for asbout 15-20 seconds.
• Tube is placed in heating block at about 95C for 20 minutes.
• Again subject it to Vortex for 15-20 seconds.
• Again centrifugation is performed at about 10,000 rpm for 2 minutes.
• Now supernatant is transferred to ependorf tube and use as DNA source.
PREPARATION OF MASTER MIX FOR PCR:
1. Arrange all reaction components on ice and prepare master mix according to table given
2. Mmix the reaction. All the liquid is collected at the bottom of the tube.
3. Capped the PCR tubes properly.
4. PCR product is obtained after complete reaction (approx. 2 hours).
5. PCR product is obtained after complete reaction (approx. 2 hours).
6. PCR product is ready for gel electrophoresis to examine the amplified bands of DNA.
Thermocycling conditions: