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Internship Report

In partial fulfillment of the requirements for the degree of

Bachelor in Microbiology

Submitted By

Muhammad Attique (Reg No 15-F-AWKUM-GCM-BS-M BIO- 07)

Adnan Ali Shah (Reg No 15-F-AWKUM-GCM-BS-M BIO- 30)

Department of Microbiolgy

Abdul Wali Khan University Mardan

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.

Thesis Approval Committee

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 ____________________

Chairman Department of Microbiology

Dr. Hazir Rahman
Associate Professor
Abdul Wali Khan University Mardan ____________________
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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 2 blood components..............................................................................................................13

Figure 3 Blood collection tubes............................................................................................................16

Figure 4 Standard formula for culturing............................................................................................19

Figure 6 autoclave with temperature setting...................................................................................20

Figure 7 Media preparation .................................................................................................................21

Figure 8 sterilization ......................................................................................................................21

Figure 10 sterilization.....................................................................................................................22

Figure 11 Gram staining ................................................................................................................24

Figure 12 Result of gram staining.......................................................................................................24

Figure 13 Zn staining .....................................................................................................................25

Figure 14 Zn T.B stain kit...............................................................................................................25

Figure 15 Slide of Zn staining .........................................................................................................26

Figure 16 hematology analyzer ......................................................................................................28

Figure 17 ESR by Westergren method ..........................................................................................31

Figure 18 cross match procedure .................................................................................................36

Figure 19 HBsAG test kit.................................................................................................................37

Figure 20 Urine sample collection sample for physical and other tests….....................................46

Figure 21: Urine strips bottles with labeled charts .......................................................................47

Figure 22: Urine strip with negative and positive results..............................................................49

Figure 23 RBC in urine under 40x objective .............................................................…………..…51

Figure 24 WBC's seen in urine sample under 40x objectives…....................................................51

Figure 25 Gel electrophoresis.........................................................................................................59

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ABBREVATIONS
ZN Zeil Neilson

CBC Complete blood picture

HB Haemoglobin

TLC Total leukocyte count

TRBC Total red blood cell

PCV Pack cell volume

HCT Haematocrit

MCH Mean corpuscular haemoglobin

MCHC Mean corpuscular haemoglobin concentration

ESR Erythrocyte sedimentation rate

HBSAG Hepatitis B surface antigen

HCV hepatitis C virus

H.PYLORI helicobacter pylori

ALT Alanine amino transferases

AST Asparte amino transferases

PCR Polymerase chain reaction

HCG Human chorionic gonadotropin

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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.

Muhammad Attique and Adnan Ali Shah

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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:

1. Acquire clinical laboratory skills.

2. Adapt and learn new procedures.

3. Operate various laboratory instruments.

4. Understand the duty and functions of the laboratory professionals.

5. Report accurate and precise results.

6. Learn how to relate tests results to patient clinical diagnosis.

The internship program is done in hospital laboratories , where students learn in supervision of
technologist/specialist/consultant.
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HISTORY OF MARDAN MEDICAL COMPLEX

Mardan Medical Complex is a well facilitated teaching hospital which is the largest health care
facility at the Mardan city of KPK. It has all the required fascilities with state of the art
diagnostic (CT scanner, MRI) and supportive services. It was established in 1997.. Mardan
medical complex has been provided with the most advanced medical technology.The location
and design of the hospital are conducive for faster recovery, better health and relief.

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:

A: Before initiation of blood sampling

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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B: Prepare procedure equipment:

1. Inspected the integrity of equipment.

2. Syringe.
3. vacutainer tubes
4. Alcohol
5. Gloves
6. Tourniquet

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.

Figure 3 Blood collection tubes

1.1. Following specimens were unable for testing:

 Labeled was uncompleted (name, age, gender, date, time of collection).

 Blood specimen hemolyzed.

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 Anticoagulated blood becomes clot by lack of mixing after collection.

 Specimen delivered to the lab out of specified time after collection.

 Specimen not stored properly until the time of testing.

 Specimen collected in anticoagulant have not proper blood anticoagulant ratio.

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MICROBIOLOGY
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2.1 COMMON MEDIA IN ROUTINE USE

I. CLED Agar (Cysteine Lactose Electrolyte Deficient): is a differential and non-
selective plating medium for the growth and enumeration of urinary tract
microorganism.

II. MacConkey Agar. It is commonly used for family enterobacteriaceae. It is a selective

medium. As bile salt does not stop the growth of enterobacteriaceae but it stops
growth of other bacteria. Bacteria that ferment lactose take a pink colour due to
production of acid. Acid turns the indicator neutral red to pink. These bacteria are
called 'lactose fermenter', e.g. Escherichia coll.

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.

Figure 6 autoclave with temperature setting

Objective:
Preparation of sterile nutrient agar for culturing.

MATERIAL AND REAGENTS:

Commercial nutrient agar, Distilled water, Scott bottles, Balance, Measuring cylinder Beaker,
Forceps, Universal bottles.

PROCEDURE
1. Required amount of broth (with agar) powder is weighed into Scott bottles and
suspended in distilled water.
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Figure 7 Media preparation

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.

2.3 GRAM STAINING:

It is microbiological technique for staining microorganism. It was developed by Dr. Christian
Gram in 1884, it helps to diffentiate bacteria on the basis of their Gram character (Gram positive
or Gram negative). Cell morphology, size, and arrangement can also be find through gram
staining.
Principle
Crystal Violet is applied on bacteria and fixed by the mordant, some bacteria are able to retain
the crystal violet and certain are washed off by alcohol. Gram positive bacteria cell wall have a
thick layer known as peptidoglycan. The action of decolorizer causes cell wall of the cell to
dehydrate, causing cell wall pores to shrink and prevents the stain from exiting the cell. So the
ethanol cannot remove the Crystal Violet-Iodine and results in blue or purple appearance of cell.
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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.

Figure 10 Gram staining

RESULT
The staining results of gram stain are as follows:
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• Gram Positive : Appears dark purple

• Gram Negative : Appears pale to dark red

Figure 11 Result of gram staining

SPUTUM EXAMINATION BY ZN STAINING:

PRINCIPLE
Zn staining is used to stain mycobacterium tuberculosis in sputum. Carbol fuschin, is a red dye
is used to stain mycobacterium. When treated with acid, the retain which is due to presence of
mycolic acid in their cell wall.

Figure 12 Zn staining
REAGENTS:
• Basic dye Carbol fuschin

• Decolorizer 20% sulphuric acid

• Malachite green
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Figure 13 Zn T.B stain kit

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.

RESULTS AND INTERPRETATION:

ACID FAST BACILLI:They appears Red in colour, straight or curved rods in
morphology, occurring singly or in small groups.
CELLS: Appear Green (malachite green)
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BACKGROUND :Appear as Green when malachite green is used.

Figure 14 Slide of Zn staining

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HEMATOLOGY
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2.1 COMPLETE BLOOD COUNT (CBC):

By hematology analyzer TLC, platelets, retics, DLC, TRBC, MCV, MCH, MPS and MCHC
were performed. These all tests collectively called complete blood count or complete picture.
This test was performed by the whole blood which was not clotted. The CP test was performed
by the hematology analyzer which automatically sucked the blood and display result within one
minute on the computer screen.

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.

Figure 15 hematology analyzer

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The CP report was computerized. In CP following tests were performed

Table 1 CBC report
Test Reference range

Hb Male: 13 - 17 g/dl
Female: 12 - 15 g/dl

RBC 4.0 - 6.0 mil/cmm

HCT Male: 42 - 50 %
Female: 37 - 57 %

MCV 77 - 97 Fl

MCH 26 - 33 pg

MCHC 31 - 36 g/dl

WBC’s 4000 - 11000 /cmm

Neutrophils 39 - 74 %

Lymphocytes 20 - 44 %

Eosinophils 1-4%

Monocytes 2-6%

Basophils 0-1%

Platelets 150,000 - 400,000 /cmm

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2.2 MANUALLY TESTS IN HEMATOLOGY LAB. ERYTHROCYTE

SEDIMENTATION RATE (ESR):
The erythrocyte sedimentation rate (ESR) is a common hematological test it is used for the
detection of nonspecific inflammation that is caused by infection.

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

Figure 16 ESR by Westergren method

2.2 SICKLING TEST:
It is used for the determination of sickle cell. Sickle cell disease (SCD) is associated with red
blood cells. People with this disease have abnormal cell shape. Instead of having normal red
blood cells, they appears as crescent moon shaped.

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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Sample Collection And Preparation:

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:

1. 0.2 grams of sodium meta-bi-sulphate dissolve in 10 ml of distal water.

2. 5 drops of this solution and 1 drop of blood then mix it well.
3. Put drop of this mixture on glass slide, place cover slip on the drop in such a manner to
avoid bubble formation
4. Cover the edge with wax/vaseline mixture.
5. Examine it under a microscope.
INTERPRETATION
1. The appearance of red blood cells show positive sickle test (Fig. 2.4).
2. If the cover slip is not sealed properly then the resuld obtained is considered invlaid.
3. We cannot differentiate sickle cell disease and sickle cell trait even the result is positive.
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SEROLOGY
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3.1 BLOOD GROUPING

Principle:
It is based on antigen and antibody reaction. Basically there are 3 types of antisera for ABO and
1 antisera for Rh D.If one type antisera is mixed with RBC’s and it results in agglutination that
means the corresponding antigen is present on the surface of RBC’s. If we mix antisera-A and
gives precipitation with red cells means the group is A. When precipitation takes place with anti-
B RBC mixture then B group. If No reaction occur on A or B then it is confirmed as O. If
positive reaction occurs upon addition of on both A & B then the group is AB .Normally, tube
test is done in blood banks. Reverse test also done to confirm the ABO group.
Requirements:
• Antisera A
• Antisera B
• Antisera C
• Test Tubes
• Slides
Forward grouping

• Tube method
• Tile method
• Slide method

Procedure:

1. Place one drop of each antisera A, B and D on three different on a slide.

2. And one drop of blood sample and mix with sterile stick.
3. Observe for agglutination.
INTERPRETATION
ABO TYPING
• Upon addition of Anti-A serum, blood precipitation shows you have type A blood
• Upon addition of Anti-B serum, blood agglutination shows you have type B blood
• Upon addition of Both anti-A and anti-B serums, blood agglutination shows you have
type AB blood
• If blood cells donot precipitate upon addition of anti-A and anti-B are added, then you
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have type O blood.

3.3 HBsAG:
PRINCIPLE:
In this we detect HBsAG in the blood serum with the help of lateral flow immunoassay. Upon
testing the plasma specimen reacts with the anti-HBsAG antibody coated particle The mixture
migrates through the membrane by capillary action to react with anti-HBsAG antibodies on the
membrane and give a colored line upon reaction. The appearance of colored line in the test
region shows a positive result.

Figure 18 HBsAG test kit

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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Figure 24: HBsAG results

3.4 Dengue
Principle:
Rapid test is a lateral flow chromatographic immunoassay.Upon sample addition to the test kit,
the mixture passes through the antibody binding/gold complex, which then binds the
immunoglobulins in the sample.when this mixture passes through antigens on the membrane, if
any antibodies to are present, the antigens will capture them in turn. This results in appearance of
pink/purple line in the T (Test) zone of the test strip.

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.

Figure 25: Dengue test results

3.5 HCV:
Principle:
It is used for the detection of HCV in Plasma or serum. Anti-HCV antigens is coated over
membrane on the test line region of test.In test the anti-HCV antigen coated particle react with
serum and give a colour line which shows positive test result.And if no colouired line appeared
the result is considered as negative.
• Assay Diluent
• Dropper
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• 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.

Figure 26: HCV device

3.2 H.PYLORI
PRINCIPLE:
It is performed to detect antibodies produced against H.Pylori performed through device.
H.Pylori antigen is suspended on membrane in the test H.pylori antigen is immobilized on the
test region of the membrane.When performing test sample taken when react with H.Pylori antibodies
and form a color line which indicate positive result and if no color line appear then result is considered
positive.
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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

1. Negative result: Formation of only one color line.

2. Positive result: Appearance of two lolored lines.
3. Invalid result: No color line is formed in test region.

3.7 BRUCELLA: (SLIDE SCREENING TEST FOR BRUCELLA ANTIBODIES)

Wright and Smith diagnosed Brucellosis for the first time by the use of tube agglutination test.
PRINCIPLE:
Patient serum is taken and mixed with a killed BRUCEL -RB antigen.If antibodies specific to
Brucella antigen is present then it will precipitate.
REQUIREMENTS:
• Blood (serum)
• Centrifuge machine
• Pipette
• Brucella Abortus Regent
• Brucella Melitensis Regent
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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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4.1 COMPLETE URINALYSIS:

We examine urine physically, chemically and microscopically
PHYSICAL CHARACTERISTICS OF URINE:
We check following charateristics of urine in physical observation.
COLOUR:
Normal urine color is light yellow.Urochrome is generally responsible for urine yellowish color.
The change in color of urine from normal is due to vegetables or drugs.
VOLUME:
1200 to 1500 ml of urine is passed by an adult in 24 hours normally.
ODOR:
In prescence of ketones bodies fresh urine has fruity odor.Urine odor is ammonical due to
presence of bacteria.
TURBIDITY:
Cloudy urine may be due to prescence of of phosphates, urates, mucus, bacteria, epithelial cells,
or leukocytes.Normall urine is clear and transparent.

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:

• It was normally of acidic (pH 6.0).

• It was measured by the strip of paper with forceps.
• Normal pH of the urine:, pH = 5.0 to 7.0
• Acidic pH 4.5 to 5.5 was occurred in diabetes mellitus, tired molecules, etc.
• Alkaline pH 7.8 to 8.0 was seen in UTI.

Figure 20: Urine strips bottles with labelled charts

Glucose:
Urine is normally free of glucose.
• Glucose was detected by strip method.
• It was examine the color change of mixture.

Table 2 interpretation of glucose

Colour Result %
Blue Negative
Green A trace
Green with yellow precipitates + 0.5%
Yellow to dark green ++ 1%
Orange +++ 1.5%
Brick red ++++ 2%
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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 ++

Heavy cloudiness +++ and above expressed as ++++

BILE PIGMENTS:

• The bile pigments were detected by the dip strip method.

• Colour production was indicating the bile pigments.

Table 4 interpretation of bile pigment in urine

Test Colour produced
Negative test Faint yellow colour

Weak positive Pale green color +

Positive test Deep green colour ++

BILE SALTS:

• By producing the colour was observed the bile salts presence.

• The colour was changed by the mixing of sodium nitroprusside in urine tube.
• After dissolving it was bile salts were present.produced the color if
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Figure 21: Urine strip with negative and positive results

Negative test No change in colour.

Purple ring at the junction of two layers.

Positive test:

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.

• It was detected by the strip.

Colour produced Result
No colour Negative
Grace +
Light pink ++
Purple +++

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.

Table 5 interpretation of blood in urine

colour produced
Result
Yellow Negative
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Yellow colour with granules +

Green ++

Dark 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.

Figure 22 RBC in urine under 40x objective

WBC’s:
WBC’s in normally very less in urine.Increase in WBC’s in urine indicate infection somewhere
in the urinary tract. If also seen with bacteria (see below), they indicate a likely urinary tract
infection. which was seen as:

• Clear granular disc was in intact WBC’s.

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• Less granular, shrunken and distorted shape was in degenerated WBC’s.

• Clumps of many degenerated cells were in case of pus.

• Presence of many WBC’s was in clumps mean infection of UTI.

Figure 23 WBC's seen in urine sample under 40x objective

Epithelial Cells
Epithelial cells are very few in normal urine.Its number icreases in urine due to urinary tract
infections or inflammation:

BACTERIA, YEAST AND PARASITES

Urine of a healthy person collected is sterile and free from microbes the prescence of any type of
microbe is indication of infection.

4.1 GLUCOSE TEST GOD PAP METHOD

Glucose test is performed to find out level of glucose whether it is higher or lower than normal
and to related it to its cause.It is used to diagnose diabetes mellitus, hyperthyroidism,
hyperactivity of the pituitary gland overproduction of insulin by the pancreas, with tumors of the
pancreas, as well as with hypofunction of the organs involved in glucose synthesis and
carbohydrate metabolism.

SAMPLE MATERIAL:
Serum heparinized or EDTA plasma.
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PROCEDURE:

• Take 100µl of reagent in a glass tube.

• Take 10µl of plasma and incubate it at 37C for 5.
• Sample tube is then loaded on the analyzer after setting the required program for glucose.
Results are displayed on screen.

URIC ACID (ENDPOINT):

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.

4.4 ALANINE AMINOTRANSFERASE TEST:

ALT is an enzyme present in hepatocytes. When cell is damaged it is released in blood.We can find out
extent of liver from amount of ALT prescence in blood.

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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machine within 3-4 sec.

• Note down the reading from machine.
REFERENCE RANGE:
Adults Women;31U/ML

Adult’s men; 41 U/L

4.1 ASPARTATE AMINOTRANSFERASE TEST

Aspartate aminotransferase is an enzyme mainly present in liver and some other body parts such
as kidney, liver and pancreas. By measuring its value in blood we can predict or find out an
infection in liver or other organs.

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:

AST concentration is from 5 to 40 IU/L normally.

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MOLECULAR BIOLOGY
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5.1 DNA EXTRACTION:

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.

Table 6 component of PCR

Component 25 μl reaction 50 μl reaction
10X Standard Taq Reaction Buffer 2.5 μl 5 μl
10 mM dNTPs 0.5 µl 1 μl
10 µM Forward Primer 0.5 µl 1 μl
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10 µM Reverse Primer 0.5 µl 1 μl

Template DNA Variable Variable
Taq DNA Polymerase 0.125 µl 0.25 µl
Nuclease-free water to 25 µl to 50 µl

Thermocycling conditions:

Table 7 Thermocycler condition

STEP TEMP TIME
Initial Denaturation 95°C 30 seconds
30 Cycles 95°C 15-30seconds
65-68°C 1 minute/kb
Final Extension 65-68°C 5 minutes
Hold 4-10°C

5.1 Agarose gel electrophoresis

1. DNA samples to be separated for desired purpose is loaded with loading dye.
2. Connect power supply to both electrodes.
3. Add running buffer in such amount that gel surface is covered.
4. Remove the lid. Slowly and carefully load the DNA samples into the gel.
5. Turn on power and allow the gel to run to such extent that allows dye migration to an
appropriate distance.
6. Upon completion of electrophoresis, power supply is turned off and the lid is removed.
7. Remove gel from the gel box. Place the gel tray on paper towels to absorb extra running
buffer.
8. Gel is subjectged to the UV. DNA bands appears as fluorescent bands. Take picture of the
gel.
9. Running buffer and gel is properly disposed .
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Figure 24 Gel electrophoresis