IDENTIFICATION METHOD USED FOR SCREENING OF PATHOGENIC

MICROORGANISM IN CLINICAL LABORATORY

Submitted by:

MAHRUKH BIBI

SUPERVISOR: [Link] ASLAM

Semester: 8th

Department of Microbiology

SHAHEED BENAZIR BHUTTO WOMEN

UNIVERSITY PESHAWAR

Session: 2018-2022

i
 SHAHEED BENAZIR BHUTTO WOMEN UNIVERSITY
PESHAWAR

IDENTIFICATION METHOD USED FOR SCREENING OF PATHOGENIC

MICROORGANISM IN CLINICAL LABORATORY

An Internship Report Submitted to the Department of MICROBIOLOGY, in partial

fulfillment of the requirements for the BS- 4 Year degree in microbiology.

Supervisor REVIEWER
NAME: [Link] ASLAM
Assistant professor NAME: _________
Signature: Signature: ____________

Countersigned by

Coordinator (Microbiology)

ii
 SHAHEED BENAZIR BHUTTO WOMEN UNIVERSITY
PESHAWAR

SUPERVISOR CERTIFICATE
Certified that Mahrukh Bibi student of the Department of Microbiology has successfully
completed their internship report entitled:

IDENTIFICATION METHOD USED FOR SCREENING OF PATOGENIC

MICROORGANISM IN CLNICAL LABORATORY
In the Hayatabad Medical Complex (HMC) under my supervision within prescribed time and
that no report on the same title has earlier been submitted by any student.

Supervisor(s)

Name: Dr. Misbah Aslam

Assistant professor (Microbiology)

Signature: _______________

Countersigned by_________________ Coordinator of Microbiology Department:

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 PREFACE

This report is the outcome of our university support in conducting our internship period at
Hayatabad Medical Complex (HMC). Three months internship period was carried out in
laboratories of Hayatabad Medical Complex (HMC) Peshawar. Hayatabad Medical Complex
(HMC) Peshawar was established in 1984. They have well-equipped laboratories and
experienced technicians. Accurate diagnosis of disease is critical; inaccurate diagnosis may lead
to false results due to which patient may not be treated properly. For this reason should know
about standard diagnostic procedures, should be well aware of all precautionary measures to be
followed to avoid wrong results. The purpose of internship is to learn the standard diagnostic
procedures of interpretation of correct result. To become aware of difficulties faced during
performance and how to overcome these difficulties. The ultimate goal is to acquire necessary
practical skills in performing various laboratory tests in different laboratory sections which will
be applied for the diagnosis which in turn will help to improve health care services.

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 ACKNOWLEDGMENT

All the praise belongs to ALLAH Almighty; the most beneficent and merciful, who gave me the
strength, knowledge, and power to complete my internship within the specified time.

I am thankful to and fortunate enough to get constant encouragement, support, and guidance
from Coordinator Dr Sadia Bhutt and all teaching staffs of microbiology department SBBWU
which helped me in successfully completing my internship and report work.

Special thanks to my supervisor Dr. Misbah Aslam for their encouragement and more over for
their timely support and guidance till the completion of my work.

I would like to extend my sincere esteems to all staff in laboratory of Hayatabad Medical
Complex (HMC).

v
 CONTENTS

PREFACE _________________________________________________________________________ iv
ACKNOWLEDGMENT ______________________________________________________________ v
LIST OF TABLES _________________________________________________________________ viii
LIST OF FIGURES _________________________________________________________________ ix
ABSTRACT_________________________________________________________________________ x
CHAPTER 1 ________________________________________________________________________ 1
INTRODUCTION __________________________________________________________________ 1
OBJECTIVES ______________________________________________________________________ 3
CHAPTER 2 ________________________________________________________________________ 4
2.1 PHLEBOTOMY SECTION ______________________________________________________ 4
2.1.1 Registration of patient _________________________________________________________ 4
2.1.2 Sample collection_____________________________________________________________ 4
2.1.3 Dispatch of samples ___________________________________________________________ 5
2.2 MICROBIOLOGY SECTION ____________________________________________________ 5
2.2.1 Preparation of Different Culture Media ____________________________________________ 6
2.2.2 Culturing of Different Samples __________________________________________________ 12
2.2.3 Staining ___________________________________________________________________15
2.2.4 BIOCHEMICAL TESTs _______________________________________________________ 17
2.2.5 ANTIBIOTIC SENSITIVITY TEST _____________________________________________ 24
2.3 SEROLOGY SECTION _________________________________________________________ 26
2.3.1 HIV test ___________________________________________________________________26
2.3.2 HCV test __________________________________________________________________27
2.3.3 Urine pregnancy test _________________________________________________________28
2.4 BLOOD BANK ________________________________________________________________ 29
2.4.1 ABO Blood group ___________________________________________________________ 29
2.5 HEMATOLOGY ______________________________________________________________ 31
2.5.1 CBC (Complete Blood Count)__________________________________________________31

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 2.5.2 Giemsa staining _____________________________________________________________ 32
2.5.3 Blood smear ________________________________________________________________ 33
2.6 CLINICAL PATHOLOGY ______________________________________________________ 35
2.6.1 Urine R/E (Urine Routine Examination) __________________________________________35
CHAPTER 3 _______________________________________________________________________37
RESULT ________________________________________________________________________ 37
3.1 Culturing of different samples ___________________________________________________37
3.2 Gram staining ________________________________________________________________ 37
3.3 Biochemical Test for Different Bacteria____________________________________________38
3.4 Antibiotic sensitivity ___________________________________________________________ 42
3.5 Serology section ______________________________________________________________ 43
3.6 Blood bank __________________________________________________________________45
3.7 Hematology__________________________________________________________________45
3.8 Clinical pathology _____________________________________________________________ 46
CHAPTER 4 _______________________________________________________________________47
Discussion ________________________________________________________________________ 47
CHAPTER 5 _______________________________________________________________________49
Conclusion _______________________________________________________________________ 49

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 LIST OF TABLES

Table 1 Normal values of blood components_______________________________________ 31

Table 2 Culturing of samples Result _____________________________________________ 37
Table 3 Result of gram staining _________________________________________________ 37
Table 4 Result of biochemical tests ______________________________________________ 38
Table 5 Result of Antibiotic sensitivity ___________________________________________ 42
Table 6 Result of HIV, HCV, HBS Test __________________________________________ 44
Table 7 Pregnancy test ________________________________________________________ 44
Table 8 Result of 1 weak tests __________________________________________________ 44
Table 9 CBC (Complete Blood Count) Result ______________________________________ 45
Table 10 Urine Routine Examination Result _______________________________________ 46

viii
 LIST OF FIGURES

Figure 1 blood agar ___________________________________________________________ 8

Figure 2 maCconkey agar ______________________________________________________ 9
Figure 3 CLED agar _________________________________________________________ 11
Figure 4 Antibiotic sensitivity test ______________________________________________ 226
Figure 5 Gram Negative (pink) and Gram Positive (purple) ___________________________ 38
Figure 6 Positive (Staphylococcus) Negative ([Link]) ________________________________ 39
Figure 7 positive (staphylococcus aureus) Negative (staphylococcus epidermidis) _________ 40
Figure 8 positive shows pseudomonas and Negative shows ([Link]) ____________________ 40
Figure 9 Red ring shows positive result, no appearance of red ring shows negative result. ___ 41
Figure 10 triple sugar iron _____________________________________________________ 42
Figure 11 HIV test ___________________________________________________________ 43
Figure 12 Shows positive result for HCV _________________________________________ 43
Figure 13 Shows positive and negative result for pregnancy test _______________________ 44
Figure 14 shows ABO blood group ______________________________________________ 45

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 Abstract

The internship of three months was performed at Hayatabad Medical Complex (HMC).
Pathological diagnostic tests were performed to analyze samples of body fluids or tissues to test
for monitoring treatment. The main objective of the internship was to get skills and to know
about the identification methods used for screening of pathogenic microorganisms in clinical
laboratory. This report reflected all diagnostics techniques that had learned in the section of
phlebotomy, microbiology, clinical pathology, hematology, serology, and blood bank in
Hayatabad Medical Complex. The diagnostic techniques performed in this study were
preparation of media, inoculation of different clinical specimens, gram staining, antibiotic
sensitivity test, identification of microorganisms , serological test (HIV, HCV, HBs and
pregnancy tests), complete blood count, Giemsa staining and blood smear. It was concluded that
the work done during that period of time has really helpful to enhance the practical skills and
knowledge about diagnosis of disease and accurate interpretation of results.

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CHAPTER. 1

Introduction

Microbiology is defined as the study of organisms too small to be seen with the naked eye. These
organisms include bacteria, viruses, fungi, algae, protozoa. They are both essential for our health
and the cause of infectious disease. Diagnostic microbiology must identify the pathogenic
microbes that cause disease. The diagnostic microbiology laboratory is essential for diagnosis
and treatment of infectious diseases. It can be performed by examining different samples of
blood, urine, stool, pus, swab, other body fluid, sputum, wound, mucus or spinal fluid. The
purpose of microbiology lab is to provide the physician with information concerning the
presence or absence of microorganisms that may be involved in the infectious disease process.

Current internship took place in Hayatabad Medical Complex Peshawar in clinical Pathology
Laboratory Department. It covers all clinical sections like Microbiology, Serology, Blood bank,
Chemistry, Special Chemistry and Hematology. During internship period I was trained in
Microbiology, Serology, Blood bank, Hematology. In these sections different techniques were
used to identify the infectious disease.

In microbiology section of pathology department different basic procedures were practically

performed to examine and characterize microbes including culture of different samples, staining,
biochemical tests, antibiotics sensitivity test. For culturing of microorganisms used different
culturing media such as Blood agar media, MaCconkey agar media (for blood, stool, pus,
sputum, and body fluid samples). CLED media (for urine sample) and Nutrient media (for
antibiotic sensitivity test). Microorganisms are identified through gram staining and biochemical
tests.

In serology section HIV, HCV, HBS, pregnancy test were performed by readymade kit which
provided by manufacturer company.

In blood bank Blood grouping and cross match were performed.

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In hematology section CBC (Complete Blood Count) which is performed by automatic analyzer
and GIEMSA staining.

This internship helped me how to apply my skills in practical field. It also enhances my practical
skills, my knowledge about disease diagnosis.

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 Objectives

The aims and objectives of this internship was:

1. To become proficient in laboratory skills and safety protocols.

2. To isolate microorganism using aseptic technique

3. To identify various infectious microorganism through culturing, staining and biochemical

techniques.

4. To become familiar with how to dispose of treated samples.

5. To determine the concentration of biochemical composition in human body.

6. To know how to perform different serological tests.

7. To interpret the antibiotic sensitivity test of clinical isolates through disc diffusion method.

3
CHAPTER. 2

Methodology

2.1 Phlebotomy Section

Phlebotomy section followed collection of samples for in vitro tests from patient, registration of
patient and dispatch of samples to respective department.

2.1.1 Registration of patient

The reception staff registered the patient and documents his/her identification and demographic
data. After that patient are required for sample collection.

2.1.2 Sample collection

Today’s technologies allow testing on wide variety of samples collected from the human body.
Most often, all that is required is a blood sample. However, samples of urine, saliva, sputum,
pus, wound, Stool, semen, and other bodily fluids and tissues also can be tested.

1. Blood collection

Venipuncture is most preferred method for blood collection. Decontaminate the area of
venipuncture than insert a needle in to a vein in the arm and draw the sample, If the sample is
drawn for blood culture then transfer the sample into culture bottle and mixed the blood with
broth, Heart brain infusion media is present in culture bottle, blood sample is then submitted to
laboratory where it is cultured. If the sample was drown for other test then transfer to other test
tubes.

2. Urine collection

Most urine specimens are collected by having the patient urinate into a container or receptacle.
To keep the sample from becoming contaminated by materials outside the urinary tract, patients
are given instructions on how to clean the genital area and void a bit of urine before collecting
the specimen into the container. The most common method for collection of urine sample is the
midstream clean-catch method. For this method we use urine cup to collect sample. Remember
to wash hands well after collecting the specimen.

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 3. Sputum collection

Patients are instructed to cough up sputum from as far down in the lungs as possible. (A health
practitioner may assist the patient in some situations.) This is best accomplished first thing in the
morning before eating or drinking, by taking several deep breaths before expectorating into the
collection cup. Sputum should be relatively thick and not as watery as seen when producing
saliva.

4. Pus and wound collection

Before collecting the sample clean the surface and around area of wound by cotton. Take a swab
for collection. If the wound is open, press the tip of a cotton swab into the wound and gently
rotate it to collect a sample. If the wound is closed, they can withdraw fluid or pus from the
wound with a syringe and a small needle. This is called needle aspiration

5. Stool collection

Patients usually collect this sample themselves during toileting, following instructions to prevent
the sample from becoming contaminated from other material in the toilet bowl. Patients may also
be told to avoid certain foods during the test period. Depending on the test, patients may be
instructed to collect the sample in a container, scoop a small portion into a vial, or smear a small
amount on special test paper. Wash hands after handling the sample.

2.1.3 Dispatch of samples

Samples are dispatched to their respective departments of laboratory.

2.2 Microbiology Section

Microbiology section is composed of four different section including bacteriology, parasitology,

serology and mycology.

Isolation and identification of microorganisms can be done by the following steps.

1. Preparation of different culture media

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 2. Inoculation of different samples
3. Gram staining
4. Biochemical tests
5. Antibiotic sensitivity test

2.2.1 Preparation of Different Culture Media

Culture media are mediums that provide essential nutrients and minerals to support the growth of
microorganisms in the laboratory.

Microorganisms have different nature, characteristics, habitat, and even nutritional requirements,
thus it is impossible to culture them with one type of culture media so we use different culture
media for growth of different microbes according to their growth requirements. However, there
are also microorganisms that can’t grow on a culture media at all in any condition these are
called obligate parasites.

Preparation of different solid media was performed including Blood agar, MacConkey agar,
CLED agar and Nutrient agar in microbiology laboratory of HMC. Basically agar is gelatinous
substances that extracted from seaweeds and made up of agarose and agaropectin. Agar is ideal
solidifying agent for culture media that provide surface area and nutrient for the growth of
microorganisms. Agar help to isolate organisms found in minority either enhance the growth of
some organisms or inhibit the growth of other organisms.

1. Blood agar media

Blood agar is enriched medium use to culture those bacteria or microbes that do not grow easily.
Such bacteria are called fastidious as they demand a special, enriched nutritional environment as
compared to the routine bacteria. Blood agar is use to grow a wide range of pathogens
particularly those that are more difficult to grow such as Haemophilus influenzae, Streptococcus
pneumoniae and Neisseria species. It also act as differential media which helps in detection of
hemolysis by cytolytic toxins secreted by some bacteria such as certain stains of Bacillus,
Streptococcus, Enterococcus, Staphylococcus, and Aerococcus.

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Components of blood agar

• 0.5% Peptone
• 0.3% beef extract/yeast extract
• 1.5% agar
• 0.5% NaCl
• Distilled water
• 5% Sheep Blood
• pH should be from 7.2 to 7.6 (7.4)

Preparation of blood agar media

1. About 40 grams of the prepared medium is added to 1000 ml distilled or deionized water.

2. The suspension is heated up to boiling to dissolve the medium completely.

3. It is then sterilized by autoclaving it at 15 lbs pressure and 121°C for about 15 minutes.

4. The medium is then taken out of the autoclaved and cooled to about 40-45°C.

5. 5% v/v sterile defibrinated blood is added aseptically and mixed well.

6. The media is then poured into sterile Petri plates under sterile conditions.

7. Once the media solidifies, the plates can be placed in the hot air oven at a lower heat
setting for a few minutes to remove any moisture present on the plates before use.

Uses of blood agar media

➢ Routine culture
➢ Use for isolation and cultivation of many types of fastidious bacteria.
➢ It is also an indicator medium showing the hemolytic properties of bacteria such as
streptococcus pyogenes.

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 Figure 1 blood agar

2. MacConkey agar media

MacConkey agar media is solid, selective and differential media that only cultivates gram-
negative bacterial species. it can further differentiate Gram-negative organisms based on their
lactose metabolism. Selective ingredients are the bile salts and the dye crystal violet which
inhibit the growth of gram positive bacteria. The media also has the ability of inhibiting the
swarming of proteus.

Composition of MacConkey agar media

• Peptone (pancreatic digest of gelatin) 17gm

• Protease peptone (meat and casein) 3gm
• Lactose monohydrate 10gm
• Bile salt 1.5gm
• Sodium chloride 5gm
• Crystal violet 0.001g
• Neutral red 0.03gm
• Agar 13.5gm
• Distal water 1 liter

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Preparation of MacConkey agar media

1. About 49.53 grams of dehydrated powder is added in 1000 ml purified/distilled water.

2. Mixed them well to form suspension.
3. Suspension is heated to boiling to dissolve the medium completely.
4. Sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
5. Allowed the media to cool to 45-50°C.
6. Mixed well
7. The media is then poured into sterile Petri plates under sterile condition.
8. Allowed them to solidify at room temperature.
9. Stored prepare media at low temperature.

Result interpretation

➢ Lactose fermenting bacteria grow as pink. The pink color is due to production of acid
from lactose.
➢ Lactose non-fermenting bacteria such as Shigella and Salmonella are colorless and
transparent. Yersinia enterocolitica may appear as small, non-lactose fermenting colonies
after incubation at room temperature.

Figure 2. MaCconkey agar

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 3. CLED agar media

CLED (cysteine-, lactose-, and electrolyte-deficient) agar is a differential culture medium

primarily used to isolate and enumerate bacteria, especially from urine samples. Support the
growth of all urinary pathogens, also inhibit the swarming of proteus. It also differentiate
between lactose fermenting and non-lactose fermenting bacteria.

Composition of CLED media

• Lactose 10.0gm/L
• Pancreatic digest of gelatin 4.0gm/L
• Pancreatic digest of casein 4.0gm/L
• Beef extract 3.0gm/L
• L-Cystine 0.0128gm/L
• Bromothymole blue 0.02gm/L
• Agar 15.0gm/L

Preparation of CLED agar media

1. About 36 grams of the dehydrated medium is added in one liter of distilled water to make
suspention.
2. Mixed them well
3. Heated to boil for one minute until the media is completely dissolved.
4. Autoclaved it at 121°C for 15 minutes.
5. Allowed the media to cool to 50°C and mixed well
6. Dispensed into sterile Petri plates.
7. When the medium is solidified, label the plates with the name of the media and the date
of preparation.
8. Stored media plates in an inverted position (to avoid excess moisture) at 2-8°C until use.

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Result interpretation

Lactose fermenting bacterial colonies appear yellow but have different colony morphology based
on size, while non-lactose fermenting bacterial colonies appear blue on CLED media. Colonies
of Gram-positive Colonies of Gram-positive cocci appear yellow but smaller in size as compared
to Gram-negative ones.

Figure 3. CLED agar

4. Nutrient agar media

Nutrient Agar is a general purpose, nutrient medium used for the cultivation of microbes
supporting growth of a wide range of non-fastidious organisms. Nutrient agar is popular because
it can grow a variety of types of bacteria and fungi, and contains many nutrients needed for the
bacterial growth. Nutrient agar media is also use for antibiotic sensitivity test.

Composition of nutrient agar media

• Beef extract (provide vitamins, carbohydrates, organic compounds, nitrogen salt) 30g
• Peptone (source of nitrogen) 5g
• Agar (solidified agent) 15g
• Nacl (maintain salt concentration in medium) 5g
• Distal water (essential for growth and reproduction of microorganisms) 1000ml
• pH 7

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Preparation of nutrient media

1. Suspended 28 g of nutrient agar powder in 1 litre of distilled water.

2. Mixed well.
3. Suspension is heated to boil to fully dissolved all components.
4. Once the nutrient agar had been autoclaved, allowed to cool but not solidify.
5. Poured nutrient agar into each plate and leave plates on the sterile surface until the agar
has solidified.
6. Once the plate solidify stored the plate 2-8 degree Celsius.

2.2.2 Culturing of Different Samples

1. Urine culture

A urine culture is a test that detected bacteria and yeast in urine. This test can find and identify
the germs that cause a urinary tract infection (UTI). Most UTIs are considered uncomplicated
and are easily treated. However, if they are not treated on time, the infection may spread from the
bladder and ureters into the kidneys. A kidney infection is more dangerous and can lead to
permanent kidney damage. In some cases, an untreated urinary tract infection may spread to the
bloodstream (septicemia) and cause sepsis, which can be life-threatening.

Materials

• CLED agar media

• Urine sample
• Wire loop
• Flame

Procedure

1. Urine was mixed by rotating urine sample cup.

2. Wire loop was sterile on the flame to kill germs on it.
3. Allowed the loop to cool.
4. A sterile loop was dip in urine sample, inoculated the loop full of sample on CLED
agar media.

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 5. Streaked agar by making primary, secondary, tertiary and zigzag streaks on the
media.
6. The sample plates are incubated for 24 hours.
7. After incubation period the growth of bacteria and yeast could be seen on CLED
media.

Result interpretation

After incubation if there is no growth on plate the test will consider negative. If the growth
appear on the plate test will consider positive. Some time may be more than one type of growth
this is due to infection in which more than one pathogen are involved.

Bacteria involves in infection mostly [Link], proteus spp, [Link], klebsilla. Efaecalis,
[Link] will appear on media.

2. Blood culture

A blood culture is a test that is used to detect foreign pathogens like bacteria, yeast, and other
microorganisms in blood that cause infection in blood, infection in blood cause by bacteria is called
bacteremia. Blood culture testing is frequently used to diagnose infections and determine if germs have
entered the bloodstream. A blood culture test identifies the specific germ causing an infection and enables
further testing to determine what type of treatment may be most effective.

Materials

• Blood agar media

• MacConkey agar media

• Sample in a culture bottle

• Wire loop

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 • Flame

• Incubator

Procedure

1. Clearly labeled each sample bottle with the name and number of Patient Keep the sample
bottle.

2. Inoculated media was incubated at 35-37C for seven days.

3. Checked samples two times every day for turbidity.

4. When the growth was present on 7 day the sample was sub cultured on blood agar and
MacConkey agar media.

5. For sub culturing we used syringe to take sample in the syringe from culture bottle and
placed one drop on MacConkey agar and one drop on blood agar.

6. After that sterile wire loop was used to make primary, secondary, tertiary and zigzag
streaks on the plates.

7. Then plates were incubated at 37C for 24 hours.

8. After incubation period when growth appeared on media then gram staining was done for
identification of pathogen.

9. Examine a gram stained colonies under microscope.

10. Look for staphylococci, Proteus specie, Neisseria specie,and [Link].

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 3. Wound and pus culture

A wound culture is a test that detects and identifies bacteria that cause infections (pathogenic) in
a wound. Any wound may become infected with a variety of bacteria. A culture helps to
determine whether a wound has become infected, which type of bacteria are causing the
infection, and which antibiotic would best treat the infection and help heal the wound..

Procedure

1. Sample containing swab is used to streak the sample on the blood and MacConkey
agar media by primary, secondary, tertiary and zigzag streaks.

2. Media plates are incubated at 37C for 24 hours.

3. After incubation growth was appeared.

4. Then colonies were gram stained for identification.

Result interpretation

Staphylococcus aureus, streptococcus pyogenes, pseudomonas aeruginosa, preteus species and

[Link] etc.

2.2.3 Staining

Staining is a process in which cells or tissues are colorized with a particular stain (chemicals or
dye) for identification of microbes that cause infection. Most biological structures are transparent
so stains are applied for better contrast between microbes.

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Gram staining

Gram staining is a method of differentiating between bacterial species in to two large groups
(gram positive and gram negative) of bacteria based on their different cell wall constituents. The
Gram stain procedure distinguishes between Gram positive and Gram negative groups by
coloring these cells pink or purple.

Reagents

• Crystal violet (primary stain)

• Gram's Iodine (mordant that fixes crystal violet to cell wall)
• Decolorize (ethanol)
• Safranin (secondary stain)
• Water

Procedure

Smear preparation

1. Take a clean slide free of contamination, Place a drop of normal saline on slide.
2. Pick colony from culture by sterile wire loop and mix with saline on slide to make smear.
3. Allow the smear to air

Heat fixation

1. Heat fixed the smear by passing the slide on the flam 3 times.
2. Allow the slide to cool for 1 minute.

Staining

Step1. Crystal violet (primary stain)

1. Add crystal violet on the smear for 1 minute.

2. Rinse the slide with tap water for 5 seconds.

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 Step2. Gram iodine

1. Add gram iodine on the smear for one minute this is an agent that fixes the crystal violet
to the bacterial cell wall.
2. Again wash the slide for 5 seconds on tap water.

Step3. Decolorize

1. Add decolorize on slide for few seconds.

2. Gently wash the slide.

Step4. Safranin (secondary stain)

1. Know add safranin on the smear and leave it for one minute on the smear.
2. Wash it with tap water and keep it to dry.

Result interpretation

If the bacteria are Gram positive, it will retain the primary stain (crystal violet) and not take the
secondary stain (safranin), it will appear purple under a microscope. If the bacteria are Gram
negative, it will lose the primary stain and take the secondary stain, causing it to appear pink
under a microscope.

2.2.4 Biochemical Tests

Biochemical tests are the tests used for the identification of bacterial species based on the
differences in the biochemical activities of different bacteria. Bacterial physiology differs from
one type of organism to another.

1. Catalase test
Catalase test is used to differentiate those bacteria that produce the catalase enzyme such
as staphylococci from non-catalase producing bacteria such as streptococci.

17
Principles

Catalase enzyme acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water.
Therefore when an organism come in contact with hydrogen peroxide bubbles of oxygen are
released, these organism are called catalase producing organism. If the bubble are not released
that organism is called non-catalase producing organism.

Materials

• Slide
• Hydrogen peroxide
• Inoculating loop
• Culture plate containing colonies

Procedure

1. Pic colony by sterile loop

2. Make smear on the slide
3. Add drop of hydrogen peroxide on the smear
4. Looked for immediate bubbles ( hydrogen gas) production on the smear.

Result interpretation

If a gas (bubbles) production takes place that indicates the presence of catalase producing
bacteria called staphylococcus aureus. The test will be positive.

If the gas is not produce it indicates the presence of non-catalase producing bacteria such as
Streptococcus and Enterococcus. The test will be negative.

➢ Bubbles appear on smear = catalase positive

➢ Bubbles not appear on smear = catalase negative.

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2. Coagulase test

Coagulase test is use to differentiate those bacteria that produce the enzyme coagulase
staphylococcus aureus from the bacteria that do not produce coagulase enzyme such as
staphylococcus epidermidis.

Principles

Enzyme coagulase converts fibrinogen to insoluble fibrin thus presence of this enzyme clots
plasma.

Therefor organisms which cause clotting of plasma or result in clump formation in the presence
of plasma are called coagulase producer, while the organisms do not produce clumps in the
presence of plasma are called non-coagulase producers.

Materials

• Plasma
• Slides
• Normal saline
• Wire loop

Procedure

1. Place a drop of normal saline on two clean slides.

2. Pick a good growth from culture specimen by using sterile wire loop.
3. Transfer the colony to saline water of each slide and make smear.
4. Add 1 drop of plasma to one of the slides and mix gently.
5. Look for clump formation within 10 seconds.

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 Result interpretation

➢ Clumping appear on slide within 10 seconds = coagulase positive (Staphylococcus

aureus)
➢ Clumping do not appear on the slide within 10 seconds = coagulase negative
(Staphylococcus epidermitis or [Link])

3. Oxidase test
Oxidase test is used to differentiate organisms that produce oxidase enzyme such as
Pseudomonas and Vibrio species from organism that do not produce oxidase enzyme
such as E. coli.

Principles

If the organism is oxidase producing, the tetramethyl-p-phenylen diamin (redox dye) is oxidized
to deep purple color.

Materials

• Oxidase reagent (contain tetramethyl-p-phenylene diamin dihydrochloride dye and

distal water).
• Filter paper
• Culture of 24 hours
• Loop

Procedure

1. Take a piece of filter paper.

2. Add 2-3 drops of oxidase reagent on the filter paper.
3. With the help of loop picked up colony from fresh growth of culture and Smear it on
filter paper at the spot, where the reagent has been added.
4. Observed the development of purple blue color within 10 seconds.

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 Result interpretation

Purple color development within 10 seconds = oxidase positive (Pseudomonas)

No color development = oxidase negative (.Enterobacteriaceae)

4. Indole test

This test is based upon the ability of certain bacteria to decompose amino acid tryptophan to
indole. This test helps in identification of [Link] and proteus etc.

Principle

Indole production is detected by kovac’s reagent which contain 4-p-dimethyl amino Benz
aldehyde , this compound react with indole to produce a red color compound.

Materials

• MIU medium
• Kovac’s reagent
• Sterile tubes
• Wire loop
• sample

Procedure

1. Add 5ml of MIU medium in a sterile tube.

2. Inoculate test organism in tube by using sterile wire loop.
3. Incubate the tube at 37C for 24 hours.
4. Add 0.5ml of kovac’s reagent to the broth culture.
5. Observe for the identification of organisms.

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 Result interpretation

➢ Positive result

Formation of red color ring on the top of media when the reagent is added in the tube.

Organisms include, Aeromonas hydrophila, Aeromonas punctata, Bacillus alvei,Edwardsiella

sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella oxytoca, Proteus
sp. (not P. mirabilis and P. penneri), Plesiomonas shigelloides, pneumotropica, Enterococcus
faecalis, and Vibrio sp.

➢ Negative result

No formation of red color ring on the top of media when the reagent is added in the tube.

Organisms include, Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp., most Bacillus
sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp., most Klebsiella
sp., Neisseria sp., Pasteurella haemolytica, Pasteurella ureae, Proteus mirabilis, P. penneri,
Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.

5. TSI (Triple sugar iron ) Test

TSI (triple sugar iron) test is a microbiological test use to determine whether a gram negative
bacilli ferments glucose and lactose or sucrose and forms hydrogen sulfide (H2S). this test is
performed for differentiation of members of the Enterobacteriaceae family from other gram-
negative rods.

Purpose

1. This test is use to detect organisms that ferment different types of sugars.
2. Detect the bacteria that degrade amino acid and produce H2S gas (gas will react Fe and
produce black precipitate between the slant and butt).

22
The medium used in this test contain

1. Three sugars (glucose, lactose, sucrose)

2. Peptone (protein has amino acid and sulfur)
3. Phenol red (as indicator sugar fermentation)
4. FeSO4 (a source of iron and sulfur)

Materials

• TSI agar slant

• Fresh Pure culture
• Straight inoculation needle

Procedure

1. Used a straight inoculation needle to pick up a well-isolated colony from the culture
plate.
2. Inoculate the TSI slant with a pure culture by streaking over the entire surface of slant
and then stabbing deep into the butt.
3. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 24
hours.
4. Examine the reaction of medium after 24 hours.

Result interpretation

➢ An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose fermentation

only.
➢ An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation of dextrose,
lactose and/or sucrose.
➢ An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation
results.
➢ Blackening of the medium: Occurs in the presence of H2

23
 ➢ Gas production: Bubbles or cracks in the agar indicate the production of gas ( formation
of CO2and H2)

2.2.5 Antibiotic Sensitivity Test

Antibiotic sensitivity test is microbiological test done to check the effectiveness of antibiotics
against bacteria and to select the best antibiotic for treatment of the infection. It is used because
bacteria may have resistance to some antibiotics. This means that antibiotics are less effective or
don't affect certain bacteria.

Purpose

1. To guide the doctor to select best antibiotic.

2. To control the use of wrong drugs.
3. To give advance treatment for drug resistant bacteria.

Principle

Antibiotic disk are used to generate a transparent zone in the agar close to disk this zone is called
zone off inhibition.

Materials

• Nutrient agar
• Sterile saline in 2ml tubes
• Antibiotic disk
• 24 hours old culture
• Inoculating loop
• Bunsen burner
• Incubator

24
Procedure

Preparation of inoculum

1. Prepare one plate for each organism and label the plates.
2. Using a sterile inoculating loop, pick up 3-5 colonies of organism from culture plate that
has been tested.
3. Suspend the colonies in 2ml of saline water tube.
4. Vortex the saline tube to create a smooth suspension.
5. Compare the turbidity with the standards.
6. Used this suspension within 15 minutes.

Plating

1. Flood the suspension in nutrient agar media rotate the plate many time and discard the
suspension from the plate.
2. Allow the media to dry.

Placement of antibiotic disks

1. Place antimicrobial disks on the agar surface with a dispenser or manually with sterile
tweezers (maximum 6 disks on a 9 cm diameter Petri dish).
2. Apply light pressure with tweezers or a sterile needle to ensure full contact of the disc
with the agar (some dispensers do this automatically).
3. Rapid incubation within 15 minutes following the deposit of the discs (beyond 30
minutes the zones of inhibition will be falsely enlarged).
4. Turn the Petri dish upside down and ideally incubate them within 15 min after depositing
the discs, without exceeding 30 min. If they are left at room temperature after depositing
the discs, the pre-diffusion of the antibiotics will generate falsely enlarged zones of
inhibition.

25
Examination of antibiotic plate

1. After 16-18 hours of incubation, examine each plate. If the plate has been streaked
satisfactorily and the inoculum concentration is correct, the resulting zones of inhibition
will be uniformly circular and there will be a lawn of confluent growth.
2. If individual colonies are apparent, the inoculum concentration was too light and the test
should be repeated.
3. Measure the diameters of the zones of complete inhibition (as judged by the naked eye),
including the diameter of the disc (6mm). Measure the areas to the nearest whole
millimeter, using sliding calipers or a ruler.

Figure 4 Antibiotic sensitivity test

2.3 SEROLOGY SECTION

Serology is the scientific study of blood serum for diagnosis and identification of antibodies in
the serum. Serological test are HIV, HCV, HBS, URINE pregnancy test.

2.3.1 HIV test

26
 An HIV test checks a sample of blood to see whether the patient is infected with HIV
(human immunodeficiency virus). HIV is a virus that destroys certain cells in your
immune system. These cells protect your body against diseases from germs, such as
bacteria and viruses, and fungi. If you lose too many immune cells, your body will have
trouble fighting off infections and other diseases. HIV is spread through contact with
blood and other body fluids from a person who has an HIV infection. This usually
happens during sex or when sharing needles or other items used to inject drugs.

Materials

• Patient sample
• Dropper
• HIV test kit
• HIV test buffer
• Centrifuge

Procedure

1. Blood sample of patient was centrifuged for 2 to 3 minutes to separate serum from
blood.
2. With the help of dropper 10µl serum was taken from test tube.
3. 2 drops of serum added into HIV kit.
4. 1 drop of HIV buffer is added in kit.
5. Result observed after 10 minutes.

2.3.2 HCV test

A blood test, called an HCV antibody test, is used to find out if someone has ever been infected
with the hepatitis C virus. The HCV antibody test, sometimes called the anti-HCV test, looks for

27
antibodies to the hepatitis C virus in blood. Antibodies are chemicals released into the
bloodstream when someone gets infected.

Materials

• Patient sample
• Dropper
• HCV test kit
• HCV test buffer
• centrifuge

Procedure

1. Blood sample of patient was centrifuged for 2 to 3 mints to separate the serum from
blood.
2. Serum was taken from sample with the help of dropper and added two drops serum in to
HCV test kit.
3. One drop of HCV test buffer added on the kit.
4. Observed result after 10 minutes.

2.3.3 Urine pregnancy test

HCG Urine pregnancy test measures the presence of the hormone human Chorionic
Gonadotropin (HCG) in human urine for early detection of pregnancy. The concentration of
HCG in a non-pregnant woman is usually 5mIU. At the time of the missed period the
concentration rises to about 100mIU. The test is sensitive to 20mIU of HCG and is capable of
detecting pregnancy as early as 7-10 days after conception.

Requirements

• Urine sample
• Urine pregnancy kit
28
Procedure

1. Open the sealed pouch and take out the pregnancy test strip.
2. Used strip rapidly.
3. Placed the test strip straight into the urine sample, making sure the arrows are pointing
down. Leaved strip in the urine for at least 8 seconds (recommend 10 seconds).
4. Removed the pregnancy test strip and placed on a dry flat surface.
5. Wait for colored bands to appear, depending on the concentration of HCG, results can be
seen in as little as 40 seconds. However to confirm a negative result the complete reaction
time 5 minutes is required.

➢ Positive result

If two colored lines appear on the test strip, the test is positive. One line may appear lighter than
the other, but they will both be the same thickness

➢ Negative result

If only one colored line appears on the test strip this is a negative result.

➢ Invalid result

If no lines appear anywhere on the test strip or if only the test line appears, the test has not
worked properly and is invalid. This is usually due to the strip not being wet enough. Repeat the
test with a new test strip ensuring that the strip is immersed in the urine sample for the full 10
seconds.

2.4 BLOOD BANK

2.4.1 ABO Blood group

ABO blood group system is the most important blood group system in human blood transfusion.

Karl Landsteiner discovered the ABO blood group system in 1901.

29
Based on the presence of antigen A and B, blood is divided into four groups: A, B, AB, and O
GROUP.

Blood group A: blood group with A antigen on RBC surface and B antibody in the serum is
called A blood group.

Blood group B: blood group with B antigen on RBC surface and A antibody in the serum is
called B blood group.

Blood group AB: if both A and B antigen are present blood group is called AB group and
does not contain antibody.

Blood group O: if both antigen are absent the blood group is called O group both A and B
antibodies are present in the serum.
Blood types are further organized by Rh factor:

➢ Rh-positive: People with Rh-positive blood have Rh antigens on the surface of their red
blood cells. People with Rh-positive blood can receive Rh-positive or Rh-negative blood.

➢ Rh-negative: People with Rh-negative blood don’t have Rh antigens. People with Rh-
negative blood can receive only blood that is also Rh-negative.

Materials

• Blood sample
• Clean glass slide
• Monoclonal Antibodies (Anti-A, B, and D)

Procedure

1. Paced a drop of blood sample on three different areas on slide.

2. Named the areas as A, B, and D.
3. Placed a drop of anti A on A area, anti B on B area and anti D on D area.
4. After few minutes observed the sample for agglutination.

30
2.4 Hematology

Hematology is the study of blood, its components and blood disorders.

2.5.1 CBC (Complete Blood Count)

A complete blood count, or CBC, is an easy and very common test that screens for certain
disorders of blood that can affect your health.

A CBC determines if there are any increases or decreases in your blood cell counts. Normal
values vary depending on your age and your gender. Your lab report will tell you the normal
value range for your age and gender.

A CBC can help diagnose a broad range of conditions, from anemia and infection to cancer.

Materials

• Blood sample in a test tube

• Hemaanalyzer machine

Procedure

1. Uncapped the test tubes that contain samples.

2. Placed test tubes in rack in a sequence.
3. Placed the rack in a hemaanalyzer machine.
4. Read result on computer.

Normal blood values

Table 1. Normal values of blood components

Blood components Normal level in men Normal level in women

RBC 4.35-5.65 ×10.e 6/µl 3.92-5.13 ×10.e 6/µl
WBC 3.4-9.6 ×10.e 6/µl 3.4-9.6 ×10.e 6/µl

31
 PLT 135-317 ×10.e3/µl 157-317×10.e3/µl
HB 13.2-16.6 g/dL 12-16 g/dL
MCV 80-96 fL 80-96 fL
MHC 27-31 pg 27-31 pg

2.5.2 Giemsa staining

Geimsa stain was primarily designed for the demonstration of malarial parasites in blood smears,
but it is also used in histology for routine examination of blood smears. It is use for fungal
scrapping, WBC, RBC, PLATELETS COUNT etc.

Reagents

• Gloves

• Slide

• Giemsa stain powder

• Distilled water

• Methanol 100%

• Patient sample

• Microscope

Preparation

Take one part of Giemsa stain powder and nine parts of distilled water and mix it.

32
Procedure

1. Prepared smear of sample on the slide.

2. Allowed the slide with smear to dry.

3. The slide was dip in 100% ethanol for 30 seconds.

4. Removed excess methanol and allowed to dry at room temperature.

5. Air-dried

6. Poured giemsa stain (one part giemsa stain 9 pats disttled water) on slide and was left it
for 20 minuts.

7. Washed the slide with distilled water.

8. Air-dried and examined under microscope.

2.5.3 Blood smear

A blood smear is a blood test used to look for abnormalities in blood cells, red blood cells, white
blood cells and platelets. The test provides information on the number and shape of these cells.
Which help doctor to diagnose certain blood disorders.

➢ Anemia
➢ Sickle cell anemia
➢ Iron deficiency anemia

Procedure
1. Blood sample was taken from patient.

33
 2. Blood was poured on slide from tube.
3. Smear prepared should be 3 cm long.
4. Slide was labeled.
5. Smear was fixed before staining.
6. Crystal violet stain was added on fixed smear for 10 minutes then washed slide.
7. Allowed for 20 minutes to dry.
8. Examined under microscope for identification of blood disease.

Result interpretation
Blood smear is normal if sample contain optimum number of cells and cells have normal
appearance.

➢ Red blood cell results that aren't normal, it may be a sign of:
• Anemia
• Sickle cell anemia
• Hemolytic anemia, a type of anemia in which the body destroys red blood cells faster
than they are replaced
• Thalassemia
• Bone marrow disorders
• Liver disease
• Cancer that has spread to the bone
➢ White blood cell results that aren't normal may be a sign of:
• Infection or inflammation
• Allergies
• Leukemia
• Bone marrow disorders

34
2.6 Clinical Pathology

2.6.1 Urine R/E (Urine Routine Examination)

Urine routine examination is urine analysis which is used to diagnose and treat various illness
including urinary tract infections, kidney disease and diabetes.

Physical examination

1. Observed volume of urine sample.

2. Observed color of the sample, normal urine color was clear to slightly hazy and yellow,
changes occur due to diet, medicine and disease.
3. Observed odor of urine sample. Normal smell is ammonia type. Sweet odor indicated
diabetes. Bad odor indicated infection.
4. PH of urine sample observed. Normal PH is acidic 5.0 - 6.5.
5. Observed turbidity urine sample. Urine appeared turbid due to the presence of pus cells,
bacteria and fats.

Chemical examination

Testing for protein, sugar, bilirubin, and other substances that may be a sign of different diseases

You may also have blood tests.

Microscopic examination

This test is performed to looks at a sample of urine under a microscope. It can see cells from
urinary tract, blood cells, crystals, bacteria, parasites, and cells from tumors.

We need this test to diagnose:

➢ Kidney disease

➢ Urinary tract infection

35
Materials

• Urine sample
• Centrifuge
• Slide
• Cover slip
• Microscope

Procedure

1. 15ml of urine sample was taken in tube.

2. Centrifuged at 1000 rpm for 3 minutes.
3. Poured the sample on a slide and covered by a cover slip.
4. Observed sample under microscope for the presence of erythrocytes, crystals, pus cells
and epithelial cells.

Result interpretation

➢ A high number of red blood cells may mean kidney disease, urinary tract infection, a
drug reaction, or cancer.
➢ A high number of white blood cells may mean infection or inflammation in urinary tract.

36
CHAPTER 3

RESULT

3.1 Culturing of different samples

Table 2 Culturing of samples Result

Patient MR Sample Growth /No Growth

22270510 Blood culture Salmonella typhi
1873132 Blood culture No growth
2349639 Urine culture Enterobacter spp
1738618 Other culture Pseudomonas
aeruginosa
2147516 Other culture [Link]
2346499 Wound swab No growth

3.2 Gram staining

Gram positive bacteria appear purple and gram negative bacteria appear pink in gram staining
procedure which is used for the isolation and identification of bacteria after culturing of samples.

Table 3 Result of gram staining

[Link] Bacteria name Color Morphology Result

1 Staphylococcus purple Cocci in bunch Gram positive
aureus
2 Strephylococcus Purple Cocci in chain Gram positive
pyogenes
3 Bacillus subtilis Purple Rods in chain Gram negative
4 Escherichia coli Pink Chain Gram negative
5 Pseudomonas Pink Single Gram negative

37
 aerogenosa
6 Acetobacter Pink Rods Gram negative

Figure 5 Gram Negative (pink) and Gram Positive (purple)

3.3 Biochemical Test for Different Bacteria

Table 4 Result of biochemical tests

Organisms Catalase Coagulase Oxidase Indole TSI

Staphylococcus + + _ _ _
aureus
Escherichia _ _ _ + _
coli
Pseudomonas _ _ +
Salmonella + _ _ _ _
[Link] + + + _ +

38
 3.3.1 Catalase test

Staphylococcus aureus show positive catalase test while Streptococcus pyogenes are catalase
negative.

Figure 6 Positive (Staphylococcus) Negative ([Link])

3.3.2 Coagulase test

Coagulase test is used to differentiate staphylococcus aureus (coagulase positive) staphylococcus

epidermis and staphylococcus saprophyticus (coagulase negative).

The bound coagulase is called the clumping factor and is detected rapidly by a slide test. The free
coagulase, in turn, is detected in the test tube as a result of the formation of a clot.

39
Figure 7 positive (staphylococcus aureus) Negative (staphylococcus epidermidis)

3.3.3 Oxidase test

➢ Positive oxidase reaction; Dark blue color appear within 30 seconds oxidase
positive (pseudomonas).
➢ Negative oxidase reaction; Absence of coloration oxidase negative ([Link]).

Figure 8 positive shows pseudomonas and Negative shows ([Link])

40
 3.3.4 Indole test

Indole test is use to differentiate members of Enterobacteriaceae family.

➢ Positive indole; Formation of red color ring on the top of the medium within seconds
of adding reagents.
➢ Negative indole; No color appear on top of medium after addition of reagent.

Figure 9 Red ring shows positive result, no appearance of red ring shows negative result.

3.3.5 TSI (triple iron sugar test

1. Alkaline slant/no change in butt (K/NC) or Alkaline slant/Alkaline butt (K/K) =

glucose, lactose and sucrose nonfermenter.(red/red)

2. Alkaline slant/acidic butt (K/A)- glucose fermentation only. (red/yellow)

3. Acidic slant/acidic butt (A/A)- glucose, lactose and/or sucrose fermenter. (yellow/yellow)

4. A black precipitate in the butt indicates production of H2S. H2S produced reacts with
ferric salt to produce black precipitate of ferrous sulfide.

41
 5. Bubbles or cracks in the tube indicate the production of CO2 or H2. Drawing of circle
around butt indicates that gas is produced by glucose and sucrose or glucose and lactose
and glucose, sucrose and lactose fermenter.

Figure 10 triple sugar iron

3.4 Antibiotic sensitivity

Table 5 Result of Antibiotic sensitivity

Sample Growth Sensitive Resistance

Blood Salmonella typhi PB, CT, AK, CN AMC, CAZ, CTX,
FEP, E, AZM,AMC
Sputum Acinetobacter PB, CT, CM, AK, C, FEP, TZP, AMC,
SCF, PGC IPM, CIP
Pus Enterobacter CT, PB, AK, C, IPM, CIP, CIT, CAZ, SCF,
TOX AMC, TZP
Urine Escherichia coli IMP, F, TGC, FOS, AMC, CPX, PZP,
CT, CN, AK, C CAZ, FEP
Wound swab Pseudomonas AK, C, TCC, PB, CT CIP, CN

42
3.5 Serology section

3.5.1 HIV Test

Figure 11 HIV test

3.5.2 HCV Test

Figure 12 Shows positive result for HCV

43
3.5.3 Pregnancy Test

Figure 13 Shows positive and negative result for pregnancy test

One red line in kit shows negative result while two lines show positive result for pregnancy test.

Table 6 Result of HIV, HCV, HBS Test

Patient MR No HIV HCV HBS

2298459 Positive Negative Negative
000344 Negative Negative Negative
572195 Negative Positive Negative
2202445 Negative Negative Positive
2248419 Positive Negative Positive

Table 7 Pregnancy test

Patient MR No Result
235390 Negative
2265262 Positive
2273963 Positive
2276158 Negative
2300803 Negative

Table 8 Result of 1 weak tests

Test name Total samples Positive Negative

HIV 350 0 350
HCV 350 5 345

44
 HBs 350 5 345
Pregnancy test 07 3 4

3.6 Blood bank

3.6.1 ABO Blood group

Figure 14 shows ABO blood group

3.7 Hematology

Table 9 CBC (Complete Blood Count) Result

Patient HB RBC WBC PLT MCV MCH

MR No
183394 9.57 g/dL 7.8 7.06 230 58.8 fL 20.6 pg
2308153 15.4 g/dL 5.07 9.73 291 88.2 fL 30.4 pg
2308150 7.2 g/dL 2.52 11.29 257 82.5 fL 28.6 pg
187178 14.6 g/dL 4.82 9.57 339 88.9fL 30.2pg

45
3.8 Clinical pathology

Table 10 Urine Routine Examination Result

[Link] Physical Chemical Microscopic

examination examination examination
1 Color dark yellow pH 6.0 Pus cells 2-3
Sugar Nil RBC 1-3
Albomin Nil Epithelial cell (+) (+)
2 Color pale yellow pH 8.0 Pus cells 2-4
Albomin Nil RBC 1-2
Protein Nil Epithelial cells (+) (+)
3 Color light yellow ph 7.5 Pus cells 13-18
protein (++) BBC 2-3
Epithelial cells (+)
4 Color pale yellow PH 7.5 Pus cells 2;3
Abomine Nil RBCs 4-6
Protein Nil Epithelial cells 2-3
Sugar Nil

46
CHAPTER 4

Discussion

During internship period different test performed in different sections of laboratory by medical
technician for detection of disease and its treatment. Pathology comprise of different laboratory
sections including Microbiology, Serology, Blood bank, Hematology and Biochemistry.

In microbiology section most samples were blood, urine, pus, wound swab, stool, throat swab
etc. Culturing of these samples were performed on different growth medium such as Blood agar,
MacConkey agar and CLED agar media for isolation of different types of bacteria. Gram
staining performed for differentiation of gram negative and gram positive bacteria. Biochemical
tests were performed for identification of bacterial species. Antibiotics sensitivity test helped
Doctors to select effective/best antibiotics for the treatment of patient. Most common species
that were identified are [Link], Stapylococcus aureus, Pseudomonas spp, Providencia spp,
Samonella, Streptococcus etc. Pseudomonas aerugenosa was most prevalent pathogen detected
in swab culture caused pneumonia, ear and wound infection. [Link] was mostly detected in urine
culture caused UTI. Staphylococcus detected in swab culture grow on blood agar cause skin and
47
soft tissue infections, also cause blood stream infections, pneumonia etc. Salmonella detected in
stool culture.

In serology section rapid tests were done for HIV, HCV, HBS and Pregnancy by using
readymade kits. Most cases of HCV and HBs were positive, caused by viruses and mostly due to
use of contaminated equipment or close contact with infected person.

In hematology section complete blood count test was done to check whole blood for level of
RBSs, WBCs, HB, and PLTs. High or low RBCs indicates kidney disease. High hemoglobin
indicates lungs, liver or kidney disease, while Low hemoglobin level suggest anemia. High
WBCs indicates blood cancer, Low WBCs shows bone marrow cancer and low PLTs count
shows viral infection such as HIV or HCV, high PLTs shows inflammation or infection.

In urine routine examination physical chemical and microscopic examination of urine were
performed for detection of different disorders, such as urinary tract infection, kidney disease,
liver disease, infection or inflammation and diabetes.

48
CHAPTER 5

Conclusion

During the internship we learnt procedure of different diagnostic tests to correctly diagnose the
disease and interpret the result. Diagnostic tests carried out in different sections of pathology
such as microbiology, serology hematology and blood bank. In microbiology section we learnt
preparation of different media, how to select medium according to the sample, how to perform
culturing, identification of growth on culture media either they are lactose fermenting or non-
lactose fermenting bacteria, gram staining for gram positive and gram negative, biochemical tests
for identification of bacterial specie, and antibiotic sensitivity test for selection of effective
antibiotics for treatment. In serology section get to know about how do diagnose HIV, HCV,
HBs, and pregnancy test by rapidly applied test. In blood bank get know how to identify blood
group and cross match. In hematology get knowledge about how to perform CBC. Also get
knowledge how to perform urine routine examination.

49