MICROBIOLOGY INTERNSHIP REPORT

By

Name:

Reg no:

Internship Report submitted in partial

fulfillment of the requirement for the degree of

BACHELOR OF SCIENCE

IN

MICROBIOLOGY

Internship Organization: Citi Lab & Research Centre

Internship Dates: - to -

DEPARTMENT OF MICROBIOLOGY

GC UNIVERSITY, FAISALABAD
 September 2024

DEDICATION
Dedicated to the unfathomable depths of love and ever strengthening prayer of my parents, my
siblings, friends and moral support of my honorable supervisor who brought me hope in order to
attain the destination.

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 DECLARATION
I (Name), (Registration No. ) hereby declare that the title Microbiology Internship Report at
Citi Lab & Research Centre and the content of Internship Report are the product of my own
training and no part has been copied from any publish source except the references & protocols. I
further declare that this work has not been submitted for award of any other degree. The university
may take action if the information provided is found inaccurate at any stage.

The work reported in this account was carried out by me under the supervision of Mr.
Muhammad, Lecturer Microbiology at Gulab Devi Educational complex, Lahore, affiliated
with Government College University Faisalabad, Pakistan. I also undertake that I will not
publish this work without the consent of my supervisor and I will be responsible for any plagiarism
in this report.

Signature of the student:

Name:

Registration No:

CERTIFICATE OF INTERNSHIP BY OORGANIZATION

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(upload here)

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 ACKNOWLEDGEMENT
It is all by the grace of Allah Almighty, the most beneficent and merciful, who enabled me to
complete my innovative internship report task. It is He, who heard us when we called on Him and
has showed His boundless blessings on me to endure and overcome the challenges faced in my
work. I am gently thankful to our honorable Chairperson of the Department, Prof. Dr.

I want to express deepest gratitude to my supervisor Mr. Muhammad, for their kind supervision,
beneficial comments, and enthusiasm, appreciation, vast knowledge and wise instruction then
assisted me in completing internship report work.

These acknowledgements would be incomplete without mentioning my parents for their endless
love, bundles of prayers, affections and motivation towards achieving my goals. I would like to
pay regards to my beloved siblings who have always been a source of inspiration for me. Sincere
thanks to all my friends for their moral support, friendship and memories.

TABLE OF CONTENTS
TITLE PAGE ………………….………………………………………………………………………………….…………………………….…. i

DEDICATION ...................................................................................................................................... ii

DECLARATION ................................................................................................................................. iii

v
CERTIFICATE OF INTERNSHIP BY ORGANIZATION ............................................................. iii

CERTIFICATE BY SUPERVISORY COMMITTEE .......................... Error! Bookmark not defined.

ACKNOWLEDGEMENT ................................................................................................................... v

LIST OF FIGURES ............................................................................................................................. vi

LIST OF TABLES ..............................................................................................................................viii

LIST OF ABBREVATIONS .............................................................................................................. ix

SUMMARY ......................................................................................................................................... xi

CHAPTER1: INTRODUCTION ......................................................................................................... 1

1.1 Justification of the training ............................................................................................................ 1

CHAPTER-2: DESCRIPTION OF INTERNSHIP ............................................................................ 2

2.1 Overview of Organization.............................................................................................................. 2

2.2 Plan of Internship Program ............................................................................................................ 2

2.3 Learning Experiences ..................................................................................................................... 3

2.3.1 Working in special chemistry department ...........................................................................3

2.3.2 Working in Microbiology department .................................................................................7
2.3.3 Working in routine chemistry department ......................................................................... 23
2.3.4 Working in histopathology department ............................................................................. 28
Chapter-3: CONCLUSION & LIMITATION .................................................................................. 35

3.1 Conclusion .................................................................................................................................... 35

3.2 Limitation of training program .................................................................................................... 35

REFERENCES ................................................................................................................................... 36

LIST OF FIGURES
Figure. 2.1 Citi Lab & Research Centre, Lahore ......................... Error! Bookmark not defined.
Figure. 2.2 Instruments used in ELISA technique .......................................................................4
Figure. 2.3 automation machines for ELISA ...............................................................................6

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Figure. 2.4 urine chemical examination by Combur strips...........................................................9
Figure. 2.5 microscopic visualization of different parameters in urine ...................................... 10
Figure. 2.6 different forms of sperms in microscopic semen analysis ........................................ 12
Figure. 2.7 illustration of gram-negative & gram-positive bacteria under the microscope ......... 16
Figure. 2.8 illustration of MTB under the microscopic .............................................................. 17
Figure. 2.9 streaking done by nichrome wire loop on agar plate ................................................ 20
Figure. 2.10 Staphylococcus bacteria growth on blood agar media plate ................................... 21
Figure. 2.11 E. coli bacteria growth on macConkey agar media plate ....................................... 21
Figure. 2.12 Neisseria Gonorrhea growth on chocolate agar media plate................................... 22
Figure. 2.13 Klebsiella Pneumoniae growth on CLED agar media plate ................................... 22
Figure. 2.14 Pseudomonas Aeruginosa growth on nutrient agar media plate ............................. 23
Figure. 2.15 (A=Beckman Coulter AU480; B=Roche Cobas c311 by HITACH) ...................... 24
Figure. 2.16 ICT test device showing results variations ............................................................ 25
Figure. 2.17 agglutination test showing results ......................................................................... 27
Figure. 2.18 grossing of different samples .................................. Error! Bookmark not defined.
Figure. 2.19 tissue processor machine for tissues dehydration and preservation ........................ 30
Figure. 2.20 embedding of tissues in molds of their sizes .......................................................... 31
Figure. 2.21 during microtomy of tissue blocks & sticking them on glass slide ......................... 32
Figure. 2.22 De-waxing or clearing of wax around the tissue section by placing glass slide on hot
plate .......................................................................................................................................... 32
Figure. 2.23 staining of racks holding slides of tissue ............................................................... 34

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 LIST OF TABLES
Table 2.1 Sequence of different samples on different agar’s media for culturing .................................... 18

viii
 LIST OF ABBREVATIONS
ACE Angiotensin-converting enzyme

ACTH Adrenocorticotropic hormone

ADA Adenosine deaminase

ANA Anti-nuclear antibody

APTT Activated partial prothrombin time

CEA Carcinoembryonic antigen

CPK Creatine phosphokinase

CRP C-reactive protein

DHEA Dehydro epi androsterone

DPX Dibutyl-phthalate polystyrene xylene

ENA Extractable nuclear antigen

ESR Erythrocyte sedimentation rate

H&E Hematoxylin & eosin

HCG Human chorionic gonadotropin

ICT Immunochromatographic test

iPTH Intact parathyroid hormone

LFT Liver function test

MH Meuller Hinton

PAS Periodic acid-Schiff

PH Potential hydrogen

PT Prothrombin time

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 RA Rheumatoid arthritis

RFT Renal function test

RH Rhesus factor

TG Tri-glyceride

TIBC/UIBC Total iron binding capacity

TSI Triple sugar iron

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 SUMMARY
A brief description of the work carried out in the laboratory during training at Citi Lab & Research
Centre is provided in this report. Four departments existed at CRC (Citi lab & research centre):
special chemistry, microbiology, routine chemistry, and histopathology. In the special chemistry
department, various automated machines were utilized, including Abbott Alinity i and Roche by
Hitachi Cobas 6000 + E411. On these machines, automated ELISA (enzyme linked
immunosorbent assay) was performed to analyze vitB12, syphilis, thyroid profile, cortisol, human
chorionic gonadotropin-beta, and free T4. Manual sandwich ELISA was also employed for anti-
tissue transglutaminase, anti-nuclear antibodies, h-pylori, ENA (extractable nuclear antigen)
profile, and anti-phospholipid. In the microbiology department, various techniques and procedures
were employed to study the isolation and identification of microorganisms from patient samples
using microscopy, staining, and biochemical tests. Microorganisms were cultivated in controlled
environments, and their growth and characteristics were observed. Antimicrobial susceptibility
testing (AST) was conducted to determine the effectiveness of antibiotics against specific
microorganisms. In the routine chemistry & hematology department, various routine tests and
procedures were utilized to analyze patient samples. Routine chemistry tests were conducted,
including; blood glucose and electrolyte analysis, liver and kidney function tests, lipid profile
analysis, complete blood count, rheumatoid arthritis factor, blood grouping, Coombs test, rh
antibody, prothrombin time, activated partial prothrombin time, fibrinogen, bicarbonate. In the
histopathology department, techniques for processing, examining, grossing, sectioning,
microtomy, staining, and diagnosing tissue samples were performed. Tissue processing
techniques, such as fixation, dehydration, and embedding, were also employed, along with staining
methods like hematoxylin & eosin and periodic acid schiff’s stain. Histopathological features of
various tissues and fluids were analyzed for cancer presence.

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CHAPTER-1: INTRODUCTION

Citi Lab and Research Centre is the top priority for quality in diagnostic and research fields. Citi
Lab is equipped with state-of-the-art instruments. It has qualified consultants, technical and
experienced management team which are devoted for upgradation of Citi Lab. It has network of
collection centers throughout the Pakistan and offer more than thousand number of tests for
diagnosis of different diseases.

The vision is to provide best diagnostic facilities within the reach of every individual all over
Pakistan. Provide cost effective, reliable and quality diagnostic testing through a qualified team.
Continuously improve our clinical lab services through education and research Ensure patient
confidentiality by maintaining highest ethical standards.

1.1 Justification of the training

According to the BS Hons in Microbiology degree, internship is made a compulsory course in the
final semester, since numerous skills need to be acquired on the clinical side. In a clinical
laboratory, internship may be beneficial for future career objectives, as students are trained to work
in labs or research centers through this experience. This training will be advantageous in
interviews, job placements, and will strengthen one's CV. Observations are enabled, fundamental
elements of experimental design are understood, and data on pathogenesis and disease control are
generated and analyzed by students through this internship. The students are prepared by the
internship for the professional and highly competitive environment ahead, where their goals and
dreams are to be achieved.

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CHAPTER-2: DESCRIPTION OF INTERNSHIP
2.1 Overview of Organization
Citi Lab and Research Centre Lahore is a renowned medical laboratory that has been providing
diagnostic services to patients since 1988. With a wide range of services, including hematology,
clinical pathology, histopathology, virology, molecular pathology. The team of experienced
doctors, radiologists, pathologists, and other workers ensures accurate test results, using the latest
technology and equipment. To facilitate patients, Citi Lab & research centre offers free home
sample collection and online reporting, making it easier for them to access their test results. The
lab has multiple branches in Lahore and other cities, increasing its accessibility to a larger
population.

Citi Lab & Research Centre in Faisal Town, Lahore is a branch of the renowned Citi Lab &
research centre network, providing high-quality diagnostic services to patients in the area as shown
in figure 2.1.

The Faisal Town branch of Citi Lab & research centre is committed to providing convenient
services, including free home sample collection and online reporting, making it easier for patients
to access their test results. This laboratory offers a comprehensive range of tests, including
hematology, clinical pathology, histopathology, virology, molecular pathology, and more. The lab
is dedicated to maintaining international standards and continually updates its techniques to stay
at the forefront of medical diagnostics.

2.2 Plan of internship program

The internship program at Citi Lab & Research Centre was scheduled from - to -

The durations for each department were as follows:

 Special chemistry:
 Microbiology:
 Routine chemistry:
 Histopathology:

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2.3 Learning experiences
a) Extensive knowledge about clinical laboratories was gained through this training. Kindness
and support were extended by colleagues. Significant assistance was provided in the lab during
the internship. As a result of this training, effective work can now be performed in any
laboratory or hospital.
b) Numerous challenges were encountered, and various tasks were accomplished during this
training. Dealing with patients and understanding machinery mechanisms and test sample
operations posed significant challenges. Difficulties were also experienced in generating
accurate reports tailored to specific patient types, but these obstacles were overcome. Personal
growth and self-improvement were achieved, enhancing future prospects.

2.3.1 Working in special chemistry department

Special chemistry involves the utilization of ELISA techniques. The causes of diseases are
determined through the analysis of various chemical constituents in different body fluids, such as
urine, blood, CSF (cerebral spinal fluid), and stool. The special chemistry tests are found to be
highly useful in determining the severity of diseases affecting multiple organs.

In this laboratory, numerous tests are performed, including vitB12, syphilis, HBsg, thyroid profile,
cortisol, HCG-beta, and free T4. Manual sandwich ELISA is also employed for the detection of
anti-tissue transglutaminase (IgA & IgG), anti-nuclear antibodies, h-pylori (IgG & IgA), herpes
(IgG & IgM), and ENA profile.

Principle of ELISA
ELISA works on the principle that specific antibodies bind the target antigen and detect the
presence and quantity of antigens binding. In order to increase the sensitivity and precision of the
assay, the plate must be coated with antibodies with high affinity. (Singh, 2022)

Manual ELISA tests conducted at CRC laboratory

 ANA(anti nuclear antibody)
 TT ( IgG + IgA)
 Anti-Gliadin (IgG+IgA)
 Anti mitochondrial antibody(AMA)

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  HEV (IgG + IgM)
 Dengue NS1
 H.pylori (IgG + igM)
 Stool for fecal (cal) protectin
 Anti-sperm Antibody
 Anti ds DNA
 Anti phospholipid
 17-OH progesteron

Equipments required for manual ELISA includes

 Eppendorf (as shown in figure 2.2 C)
 Pipette (as shown in figure 2.2 B)
 Centrifuge
 Automatic microplate washer
 Vortex mixture
 Microtiter wells (as shown in figure 2.2 A)
 Spectrophotometer
 Incubator (as shown in figure 2.2 E)
 Kits of specific test (as shown in figure 2.2 D)

A B C D E

Figure 2.1 instruments used in ELISA technique showing

A=Microtiter wells plate; B=Pipette; C=Eppendorf; D=ELISA test kit; E=Aerobic incubator

General procedure of ELISA

 The plate is prepared and a known quantity of capture unlabeled monoclonal antibodies are
added to the wells and incubated.

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 The antigen containing sample is then added to the plate.
 The plates are washed to remove unbound molecules.
 The primary antibodies are then added and incubated with the antigens.
 The plate is washed to remove unbound primary antibodies.
 Secondary antibody (the enzyme conjugated antibody) with avidin horseradish peroxidase
(HRP) or alkaline phosphatase (AP) is added and incubated.
 The streptavidin labeled enzyme is added; it binds to the biotinylated detection antibody.
 The plates are washed so the unbound enzyme linked antibodies are removed.
 Chromophore substrates are added and incubated and it changes to a blue color depending
on the amount of bound analyte substrate to wells. Enzyme converts substrate to detectable
product.
 Stop solution containing an acid (sulfuric acid) is added which terminates the reaction and
the color changes to yellow to wells for enzymatic reaction.
 Measure absorbance of wells using a microplate reader & read at 450nm. Absorbance
directly proportional to amount of antigen present. (Biochain Institute Inc., n.d.)

Automation tests conducted at CRC laboratory

 Thyroid profile
 C-peptide
 Adrenocorticotropic hormone (ACTH)
 Carcinoembryonic antigen (CEA)
 Beta-HCG
 Dibutyl-phthalate polystyrene xylene (DHEA)
 Cortisol
 Torch profile
 Free T3+T4
 Vitamin B12
 Anti-CCP
 Syphilis
 Progestron

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  NT-proBNP
 FOLATE
 Intact parathyroid hormone (iPTH)

Automation machines include

 Roche by Hitachi (Cobass 6000)

This analyzer run on Electrochemiluminiscence principle as shown in figure 2.3B
 Alinity I by (abbott)
This analyzer run on Chemiluminiscence principle as shown in figure 2.3A

A B

Figure 2.2 automation machines for ELISA showing

A=Alinity i by Abott; B=Roche 6000 by HITACHI

Procedure of automation ELISA

Pre-analytical steps
 Prepare patient samples according to laboratory protocols.
 Label samples with unique identifiers.
 Load samples onto the Alinity i system's or Roche Hitachi (Cobass E411) or Roche Hitachi
(Cobass 6000) sample tray.

Analytical steps

 Place the sample tray into the analyzer, system scans the tray and identifies the samples.
 Load required reagents into the analyzer's reagent compartment, the system checks reagent
expiration dates and levels.

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  Select the desired tests using the analyzer's touchscreen interface, the system checks for
any conflicts or incompatibilities.
 The analyzer retrieves a sample from the tray and processes it, the system performs the
necessary testing, including incubation, washing, and detection.

Post-analytical steps

 The analyzer calculates the test results based on the measured signals, results are displayed
on the touchscreen interface.
 Automated calculation of sample absorbance values, conversion to concentration units
(e.g., ng/ml), and determination of results (e.g., positive/negative).
 Automated verification of sample results against predefined criteria (e.g., cutoff values,
reference ranges).
 Automated generation of test reports, including patient demographics, test results, and
interpretive comments.

2.3.2 Working in microbiology department

In the microbiology department, hands-on experience was gained in various techniques and
procedures used for the isolation and identification of microorganisms from patient samples and
environmental sources through microscopy, staining, and biochemical tests. Microorganisms were
cultivated in controlled environments, and their growth and characteristics were observed.
Antimicrobial susceptibility testing (AST) was conducted to determine the effectiveness of
antibiotics against specific microorganisms. A microbiology laboratory is devoted to the culturing,
examination, and identification of microorganisms, including bacteria, fungi, yeasts, etc. Effective
infection prevention and control (IPC) relies heavily on the crucial role played by the microbiology
laboratory. The surveillance, treatment, control, and prevention of nosocomial infections are
facilitated by the important role played by the microbiology laboratory.

Samples with assigned lab numbers are labeled on test tubes. Samples are processed into various
tubes, including; urine, sputum, fluid, stool etc.

Samples received for microbiological analysis

 Urine

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  Pus
 Stool
 Sputum
 Plural fluids
 CSF
 Wound swab
 Semen
 Vaginal swab
 Blood
 Bronchial washing
 Knee joint fluid
 Catheter tips
2.3.2.1 Urine complete analysis

A urinalysis is a test that is performed on urine. It is used for the detection of wide range of
disorders, such as urinary tract infections, kidney disease, and diabetes. Urinalysis involves the
examination of the appearance, concentration, and content of urine. For instance, the appearance
of urine can be affected by urinary tract infections, causing it to appear cloudy instead of clear.
Increased levels of protein in urine are indicative of kidney disease, as they can be detected through
urinalysis. (Urinalysis - Mayo Clinic, n.d.)

Physical examination of urine

First of all, the samples were examined physically its color, smell etc.

 Color
 Appearance
 Deposit

Chemical examination of urine

For chemical examination of urine dipped the Combur strip in urine for few seconds and observed
the color of every parameter as shown in figure 2.4.

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  PH
 Specific gravity
 Protein
 Glucose
 Bilirubin
 Urobilinogen
 Blood
 Nitrite
 Ketone
 Leukocyte

Figure 2.3 urine chemical examination by Combur strips

Microscopic examination of urine

On a glass slide, urine drop is placed and a cover slip is placed over it and observe under the
microscope at 40X. Following parameters are commonly observed under the microscope:

 Pus cell/WBCS (as shown in figure 2.5A)

 RBCs (as shown in figure 2.5A)
 Casts (as shown in figure 2.5 C+D)
 Epithelial cells

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  Crystals
 Bacteria
 Mucus

A B C D

Figure 2.4 microscopic visualization of different parameters in urine showing

A=RBC’s + WBC’s, B=granular cast, C=WBC’s cast, D=epithelial cast

2.3.2.2 Stool analysis

A stool routine test is utilized for the diagnosis of various medical conditions. Gastrointestinal
discomfort or pain often necessitates undergoing this test. Medical conditions such as colon cancer,
inflammatory bowel disease, and other long-term ailments can be monitored and diagnosed
through this test. (Stool Routine Test, n.d.)

Physical examination of stool

First of all, the samples were examined physically its color, smell etc.

 Color
 Consistency
 Mucus
 Worms
 PH
 Odour

Procedure for microscopic examination of stool

 Add normal saline in 2 tubes then picked the stool sample through the stick and mixed
in both tubes.
 Now cover the tubes with the cap and mix gently.

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  By using the gauze piece, filter the solution of sample with N/S (normal saline) in
another tube.
 Centrifuge both tubes for 5 minutes. Add formalin and again centrifuge it.
 Now add few drops of ether and again centrifuge it.
 Prepare the slide and observed under the microscope (pus cells, parasite/ovacyst).
2.3.2.3 Semen analysis

A semen analysis, also called a sperm count, measures the quantity and quality of semen and
sperm. A semen analysis is used to find out if a problem with semen or sperm may be causing
infertility. The test may also be used to see if a vasectomy has been successful. (Semen Analysis,
n.d.)

Gross examination of semen

Firstly, note duration of abstinence, liquefaction time of semen sample.

 Appearance
 Consistency
 Volume
 PH

Sperm motility reported as

a) Rapid progression
b) Slow progression
c) Non-progressive motility
d) Immotile

Morphological changes

In cases of abnormality, various atypical sperm forms are observed, described, and illustrated
below as shown in figure 2.6.

 Normal & Abnormal

 Head & Tail defects
 Neck & mid piece defects

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  Cytoplasmic droplets
 Headless pinhead
 Pus cells

Figure 2.5 different forms of sperms in microscopic semen analysis

2.3.2.4 Sputum analysis

A routine sputum test is used to detect any harmful bacteria, fungi, or viruses that may be present
inside your lungs. Such a bacterial, fungal, or viral infection can not only cause severe discomfort
in your airways but could also lead to the onset of other severe complications if the infectious germ
is potent enough. (Sputum Culture, n.d.)

Procedure for microscopic examination of sputum

 Take test tube and pour sample in the tube.

 Add bleach in the same tube than gently mix and centrifuge it for 15 minutes at
2000Rpm at 15Lbs pressure.
 Discard supernatant part and make smear with the pellet & heat fix it.
 ZN staining was performed on smear & observed under the microscope at 100X at
oil immersion.

Results
Microscopic examination of AFB shows pink-stained organisms in curved, chain, or elongated
forms.

2.3.2.5 Fecal occult blood

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A qualitative method for detection occult blood in the stool. It aids in detecting asymptomatic
gastrointestinal conditions, such as colorectal cancer, polyps’ anemia and diverticulitis.

Requirements

 Hema screen slide (a special electrophoresis paper impregnated with natural guaiac
resin) contain both positive and negative performance standards.
 Hema screen developing solution (a stabilized mixture of hydrogen per oxide less
than 6% and 75% denatured ethyl alcohol in aqueous solution).
 Applicator wooden stick.

Procedure

 Compare the data of patient slip with the sample.

 With applicator, apply very thin smear of stool inside oval from the different
portion of stool.
 Apply 2 or more drop of hema screen developing solution to exposed test paper.
 Add I drop of developer directly onto control area. Read results within 30 seconds.

Results

 Blue color appears in test area ………………. Positive occult blood test
 Blue color appears in control area …………… Negative occult blood test
2.3.2.6 Test for bile pigments

Performed to detect the presence of bile pigments in the given sample of urine.

Apparatus

Test tubes, Fauchet's reagent, 10% Hack: (barium chloride), acetic acid, filter paper

Procedure

This is the most sensitive and best test for bile pigment. If the urine is neutral or alkaline, acidify
it with 2 drops of acetic acid. Take 5 ml of acetic acid (CH3COOH) in urine test tube. Add 5ml of
10% barium chloride solution. Mix well and filter for the precipitate, add one drop of Fauchet's
reagent to the precipitate on the filter paper.

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Results

If bile pigment is present, a green or blue color will be form.

2.3.2.7 Oxidase test

The oxidase test detects the presence of a cytochrome oxidase system that will catalyze the
transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-
phenylene-diamine. The dye is reduced to deep purple color. This test is used to assist in the
identification of Pseudomonas, Neisseria, Alcaligenes, Aeromonas, Campylobacter, Vibrio,
Brucella and Pasteurella, all of which produce the enzyme cytochrome oxidase. (Oxidase Test-
Principle, Uses, Procedure, Types, Result Interpretation..., n.d.)

Procedure

 Obtain a pure culture of the bacterium to be tested.

 Use a sterile inoculating loop to pick up a small amount of the bacterial culture.
 Rub the loop against the surface of a dry, sterile oxidase test strip or plate.
 Wait for 1-2 minutes to allow the reaction to occur & observe the color changes on the
strip or plate.

Results

 Positive result: The appearance of a deep purple or blue color within 1-2 minutes
indicates that the bacterium produces cytochrome c oxidase and is oxidase-positive.
 Negative result: No color change or a light pink color indicates that the bacterium does
not produce cytochrome c oxidase and is oxidase-negative.

Interpretation

 Oxidase-positive bacteria: Pseudomonas aeruginosa, Neisseria gonorrhoeae,

Neisseria meningitidis
 Oxidase-negative bacteria: Escherichia coli, Staphylococcus aureus, Bacillus subtilis
2.3.2.8 Gram Stain technique

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Gram staining is a common technique used to differentiate two large groups 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 red or violet. Gram positive bacteria stain violet
due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal
violet these cells are stained with. Alternatively, gram negative bacteria stain red, which is
attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the
decoloring process. (Gram Staining, n.d.)

Requirement

 Crystal violet stain

 Lugol's iodine
 Acetone-Alcohol decolorizer
 Safranin
 Glass slides
 Microscope

Procedure

 Fix the dried smear & cover the fixed smear with crystal violet stain for 60 seconds.
 Rapidly wash off the stain clean water.
 Tip off the water and cover the smear with lugol's iodine for 60 seconds.
 Wash off the iodine with clean water.
 Decolorize with acetone alcohol.
 Wash immediately with clean water.
 Cover the smear with safranin for 1 minute.
 Wash off the stain with clean water.
 Wipe the back of slide clean and place it in a draining rack for the smear to air dry.
 Examine the smear microscopically, first with the 40x objective to check the staining and
to see the distribution of material and with the oil immersion objective to report the bacteria
and cells.

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 Figure 2.6 illustration of gram-negative & gram-positive bacteria under the microscope

Results
Some possibly seen microorganism or cells under the microscope as given below;

 Gram positive bacteria …………. Dark purple (as shown in figure 2.7)
 Yeast cells ……………………… Dark purple
 Gram negative bacteria ………… Pale & dark red (as shown in figure 2.7)
 Nuclei of pus cells ……………… Red
 Epithelial cell …………………. Pale red
2.3.2.9 Ziehl-Neelsen technique

The Ziehl–Neelsen stain is also known as acid-fast stain and is performed in case of
suspected tuberculous meningitis. It detects mycobacteria as bright red filamentous structures,
which would otherwise stain poorly by MGG or Gram. (Ziehl-Neelsen Stain - an Overview |
ScienceDirect Topics, n.d.)

Requirement

 Carbol fuchsin stain (filtered)

 Acid alcohol 3% V/V
 Methylene blue 5g/v
 Glass slides
 Microscope

Procedure

 Heat fixes the dried smear & cover the smear with carbol fuchsin stain.

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  Heat the stain until vapour just begins to rise (i.e. About 60°C). Don't over heat. Allow
the heated stain to remain on the slide for 5 minutes.
 Wash off the stain with clean water.
 Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently
decolorized i.e. Pale pink.
 Wash well with clean water.
 Cover the smear with methylene blue stain for 1-2 minutes, using longer time when the
smear is thick.
 Wash off the stain with clean water.
 Wipe the back of the slide clean and place it in a draining rack for the smear to air dry.
 Examine the smear microscopically using the 100x oil immersion objective.

Figure 2.7 illustration of MTB under the microscopic

Result

Acid fast bacilli (AFB) appear pink in color, exhibiting curved, chain-like, or variable length rod
shapes as shown in figure 2.8

Reporting for sputum smear

If negative then ………… No AFB seen.

If positive than:

 1-9 AFB/ More than 10 AFB/field ………………. Report +++

 1-10 AFB/field ………………………Report++

17
  10-100 AFB/100 fields ………………………Report+
 100 fields ……………………………Report the exact number

Agar’s media used in microbiology department

 Blood agar
 Chocolate agar
 MacConkey agar
 Muller Hinton agar
 Cled agar

The special microbiology section performs cultures and susceptibilities on various clinical
samples. A microbiology laboratory is a laboratory devoted to the culturing, examination, and
identification of microorganisms including bacteria, fungi, yeasts, etc. The microbiology
laboratory has a crucial role in effective infection prevention and control (IPC).(What Are the Key
Activities in a Microbiology Laboratory?, 2015)

Table 2.1 Sequence of different samples on different agar’s media for culturing

Samples for culturing sequence of samples for culturing on different agars

Blood culture Add 10cc Blood in Tryptic soy broth (aerobic) & Thiosulphate
broth (anaerobic)

Blood Agar + MacConkey Agar

Csf culture Blood agar + Chocolate agar + MacConkey agar

Ear swab Blood agar + MacConkey agar + Chocolate agar

Eye swab Blood agar + Chocolate agar + MacConkey agar

Fluids Blood agar + MacConkey agar + Chocolate agar

Hvs / cervical swab Blood agar + MacConkey agar + Chocolate agar

Mouth swab Blood agar + MacConkey agar + Chocolate agar

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 Nasal swab Blood agar + MacConkey agar + Chocolate agar

Pus swab Blood agar + MacConkey agar + Chocolate agar

Skin swab Blood agar + MacConkey agar + Chocolate agar

Sputum culture Blood agar + MacConkey agar + Chocolate agar

Stool culture MacConkey agar + SS or XLD or DCA agar

Throat swab Blood agar + Chocolate agar + MacConkey agar

Urine culture CLED agar + Blood agar + MacConkey agar

Agar used for AST (Antimicrobial susceptibility testing)

 MH Agar / Nutrient Agar

Agar used for biochemical testing

 Citrate agar
 Urease agar
 TSI agar
 Indole agar
 DNase test

Instruments and equipment’s for culturing

 Microscope
 Incubator
 Micropipette
 Nichrome wire loop
 Bunsen burner
 Petri dishes
 Glass slides
 Cotton swabs

19
  Beaker & flasks
 Gloves

Procedure

 Respective sample contains a specific lab no. Then the specific sample of patient is streaked
on culture media plates as shown in figure 2.9.
 The plates are incubated at 37 ºC for 24-48 hours for bacterial growth. On next day the
plates are observed & reading is taken. Then from each plate isolated colony is picked
respectively and spread on MH agar.
 By using cotton swab spread it on entire media.
 Apply AST and specific antibiotics are picked and placed on each plate respectively.
 Antibiotics varies with organisms. Then incubate at 37 ºC.
 Next day zones are observed either sensitive, resistant or intermediate.
 The isolated colony is also streaked on TSI and citrate for its biochemical identification.
 After 24 hours incubation at 37 ºC. The organisms are identified through biochemical
identification.

Figure 2.8 streaking done by nichrome wire loop on agar plate

Results

On blood agar (BA)

 Staphylococcus aureus: white/yellow colonies with hemolysis (breakdown of RBC’s) as

shown in figure 2.10.
 Streptococcus pyogenes: white/gray colonies with hemolysis.

20
  Bacillus subtilis: white/cream colonies with hemolysis.
 Escherichia coli (E. Coli): no growth or poor growth.

Figure 2.9 Staphylococcus bacteria growth on blood agar media plate

On macConkey agar (MAC):

 E. coli: pink colonies (lactose fermentation) as shown in figure 2.11.

 Staphylococcus aureus: no growth or poor growth.
 Pseudomonas aeruginosa: no growth or poor growth.
 Salmonella Typhi: colorless colonies (no lactose fermentation).
 Klebsiella pneumoniae: pink colonies (lactose fermentation).

Figure 2.10 E. coli bacteria growth on macConkey agar media plate

On chocolate agar (CHOC)

 Hemophilus influenzae: small, grayish colonies (requires X and V factors).

 Neisseria gonorrhea: small, grayish colonies (requires X & V factors) as shown in figure
2.12.
 Staphylococcus aureus: no growth or poor growth.

21
  E. coli: no growth or poor growth.
 Pseudomonas aeruginosa: no growth or poor growth.

Figure 2.11 Neisseria Gonorrhea growth on chocolate agar media plate

On CLED agar (Cysteine Lactose Electrolyte Deficient Agar)

 E. coli: yellow colonies (lactose fermentation).

 Staphylococcus aureus: no growth or poor growth.
 Pseudomonas aeruginosa: no growth or poor growth.
 Klebsiella pneumoniae: yellow colonies (lactose fermentation) as shown in figure 2.13.

Figure 2.12 Klebsiella Pneumoniae growth on CLED agar media plate

On nutrient Agar (NA)

 E. coli: white/gray colonies.

 Staphylococcus aureus: white/yellow colonies.
 Bacillus subtilis: white/cream colonies.
 Pseudomonas aeruginosa: green/blue colonies as shown in figure 2.14.

22
  Salmonella Typhi: white/transparent colonies.

Figure 2.13 Pseudomonas Aeruginosa growth on nutrient agar media plate

2.3.3 Working in routine chemistry department

In the routine chemistry, hands-on experience was gained in various routine tests and procedures
used to analyze patient samples. Routine chemistry tests were conducted, including; blood glucose
and electrolyte analysis, liver and kidney function tests, lipid profile analysis, complete blood
count (CBC), RA factor, blood grouping, HIV screening, ADA, Coombs test, RH antibody, PT,
APTT, fibrinogen, bicarbonate etc.

All instruments & equipments include

 Micropipette
 Beckman coulter AU480 (as shown in figure 2.15A)
 JOKOH-EX-DS
 Microlab300
 Roche Cobas C311 (HITACHI) (as shown in figure 2.15B)
 Helena C4
 Microscope
 Bio-RAD variant II
 Sysmex
 ESR stand
 Test tubes
 Eppendorf
 Glass slides

23
 A B

Figure 2.14 automation machines for ELISA showing

A=Beckman Coulter AU480; B=Roche Cobas c311 by HITACH

Tests performed at routine chemistry lab of CRC

2.3.3.1 ICT/ screening tests

2.3.3.2 Agglutination/Titre tests
2.3.3.3 Automation tests

2.3.3.1 ICT/Screening tests

Immunochromatography is a testing method for detecting a disease by dropping the sample

containing an analyte onto a test strip. This is a speedy and simple technique that produces
diagnostic results in 10 to 15 minutes after dropping the sample.(Immunochromatographic
Technique | Immunochromato Reader (Lateral Flow Reader) | Hamamatsu Photonics, n.d.)

Following tests are performed by ICT method that are enlisted below:

 Beta-HCG
 HBsAg
 HCV
 HIV
 Anti H-pylori
 Multi-drug screen test
 Trop-T

Requirements for ICT tests

24
  Test strip/kit
 Sample (whole blood, serum, plasma, urine)
 Dropper

Procedure for ICT tests

 Collect a whole blood, serum, urine or plasma sample from the patient.
 Add 1-2 drops of sample to the sample buffer. Mix well.
 Remove the test strip from the foil pouch.
 Apply 1-2 drops of the prepared sample to the sample pad.
 Wait for 15-20 minutes.

Results for ICT tests

 Positive: Two pink lines appear (control line and test line).
 Negative: Only one pink line appears (control line).
 Invalid: No lines or a faint test line appears.

Figure 2.15 ICT test device showing results variations

2.3.3.2 Agglutination/Titre tests

Agglutination tests which refers to the clumping of particles together, is an antigen-antibody

reaction that occurs when an antigen (i.e., a molecule capable of triggering the adaptive immune
response) is mixed with its corresponding antibody at a suitable pH and temperature. These
antibodies are called agglutinins because they bind multiple antigens together and form a lattice-
like structure seen as clumping by the naked eye. (Agglutination, n.d.)

25
Following tests are performed by agglutination/titre method that are enlisted below:

 Direct/Indirect Coombs
 Widal test
 Blood grouping
 RA factor
 ASO titer
 RH antibody

Requirements for agglutination tests

 Glass slides
 Patient serum/plasma sample
 Positive/Negative control sera
 Dropper

Procedure for agglutination tests

 Take glass slide & add a few drops of the antigen (e.g., latex particles sera) on the glass
slide.
 Add a few drops of the patient’s serum or a known antibody solution to the glass slide.
 Mix the content of the glass slide with dropper.
 Observe the glass slide for agglutination (clumping) of the antigen.

Results for agglutination tests

 Positive result: Agglutination/clumping is observed as shown in figure 2.17

 Negative result: No agglutination/clumping is observed as shown in figure 2.17

26
 A B

Figure 2.16 Agglutination test showing results

2.3.3.3 Automation tests

A variety of chemistry analyzers exist for performing routine analysis, including sample-based and
random-access instruments with a wide range of throughput capabilities. Representative analyzers
based on dry and liquid reagent technology are discussed.(Lifshitz & De Cresce, 1988)

Following tests are performed by automation method on different types of machines that are
enlisted below:

a) Beckman coulter AU480 & Roche Cobas C311 (HITACHI)

 LFT
 RFT
 CPK
 TG
 Random + Fast sugar test
 CA
 AMY-CN
 CRP-N
 Iron
 TIBC/UIBC
 Phosphate
b) JOKOH-EX-DS
 Serum electrolyte
c) Microlab300

27
  Lactate
 Ammonia
 Aldolase
 ACE
 HCO3
 Copper
 Zinc
d) Helena C4
 PT
 APTT
 Fibrinogen
 Lupus
 D-Dimer
 Protein-C

Requirement for automation test

 Pipette
 Serum cups
 Centrifuge
 Reagents of specific test

Procedure for automation test

 Collect blood & centrifuge it to separate serum or plasma from patient sample.
 Load Specific test reagents onto analyzer using barcode scanner or manual entry.
 Add 200μl sample into the serum cup and load sample onto analyzer.
 Select test menu on analyzer & performs tests and calculates results using photometric or
enzymatic methods.
 Print or display results on analyzer.

2.3.4 Working in histopathology department

Introduction

28
Compounds of three greek words/ Histo mean tissue, Patho means Suffering, & logia means study
of. Its refer to the microscopic examination of tissue in order to study the manifestation of disease.

Histopathological instruments & equipments used at CRC

 Microtome
 Trimmer
 Hot air oven
 Hot plate
 Grossing hood
 Tissue processor
 Molds & Casettes
 Paraffin wax
 Scalped, disecting set
 Tissue floatation bath
 Equipment for embedding and vacuum
 Container for holding specimen

Steps of working in histopathology department

2.3.4.1 Grossing
2.3.4.2 Tissue processing
2.3.4.3 Embedding
2.3.4.4 Microtome sectioning
2.3.4.5 Clearing / de waxing
2.3.4.6 Staining
2.3.4.7 Mounting

2.3.4.1 Grossing

Samples of biopsy preserved in formalin are collected for diagnosis. Gross examination is the first
step in histopathology which is performed by doctors. Specific portion from the sample to be
examined is taken or sometimes whole sample of biopsy is taken which is then processed as shown
in figure 2.18. Sample or portion of sample to be processed is taken in cassettes after gross
examination. Preservative is formalin which prevents the autolysis of sample.

29
2.3.4.2 Tissue processing
The next step to grossing is tissue processing which is completed after processing in various
reagents. It is 18 hours long process. This respective tissue processor contains 12 jars as shown in
figure 2.19.

 First and second jar comprises formalin which helps in fixation. Specimens for processing
are placed in each jar for 1.5 hours after it the specimen are automatically shifted to next
reagent which is alcohol.
 Third, fourth and fifth jar contains alcohol. Time for specimen in each jar is 1.5 hour. The
function of alcohol is dehydration of tissues.
 Sixth and seventh jar contains acetone. Contact time with acetone for each jar is also 1.5
hours. It is also a dehydrating agent.
 Eight and nineth jar contains xylene. Contact time for each jar is 1.5 hours. Xylene is a
clearing agent for wax.
 Tenth, eleveth and twelfth jar contains paraffin wax. Jars of wax are placed in thermostat
at almost 58 ºCs so that wax may remain in molten state. Melting temperature of wax is 55
to 58 ºC.

Figure 2.17 Tissue processor machine for tissues dehydration and preservation

2.3.4.3 Embedding

30
  After the tissue processing next step is embedding. Specimens are placed on thermal
console whose temperature is beyond the melting point of wax (almost 67 ºC) as shown in
figure 2.20.
 Molds of different sizes are available. Molten wax from main console is filled into the
molds. Temperature of main console is almost 70 ºC. Specimens are placed in molds filled
with wax and their respective capsule cover is placed on the mold.
 After it the mold is placed on cryo console whose temperature is -11 ºC. When the blocks
are cold these are removed from the mold.

Figure 2.18 embedding of tissues in molds of their sizes showing

A=placing tissue in molds; B=arrangement of tissues in cassettes

2.3.4.4 Microtome sectioning

The blocks are being cut by microtome which is operated automatically and can be operated
manually. When microtome is initially operated it trims at 48 microns. After slicing when tissue
is exposed the trimming size is being set on 5 micron. This thin sized slice is taken and is placed
in water bath whose temperature is 54 ºC. The wax expands in hot water and tissue is taken on
glass slide (blotting) as shown in figure 2.21.

31
 B C

Figure 2.19 during microtomy of tissue blocks & sticking them on glass slide showing

A=cutting of tissue block; B= showing tissue block attach for trimming; C=tissue section sticked on slide

2.3.4.5 Clearing / de-waxing

The slides rack with blotted tissues are placed in hot air oven which is operated at 94 ºC. De waxing
takes place here. When wax starts melting it the slides rack is then placed in Jar 1 of xylene for
almost 5 minutes and then in jar 2 of xylene. Wax is dissolved by xylene as shown in figure 2.22.
Xylene works efficiently when it is placed at high temperature and it is clearing agent of wax.

Figure 2.20 De-waxing or clearing of wax around the tissue section by placing glass slide on hot plate

2.3.4.6 Staining
After de waxing staining is proceeded. In routine we perform H&E and PAS staining. Its protocol
is given below:

H&E (hematoxylin & eosin) staining

32
  Place the slides rack in alcohol 1 for 30 second, than dip slides in running tap water for 1-
2 minutes.
 Place in hematoxylin stain for 5 to 15 minutes depends upon the efficacy of stain. Place
in running tap water for 1-2 minutes
 Dip in acid alcohol which is a decolorizer and place it in running tap water for 1-2
minutes (hematoxylin is a basic dye which will stain nucleus after adding in acid alcohol
the stain on slide other than nucleic region will be removed).
 Dip in ammonia and place it in running tap water for 1-2 minutes (ammonia will turn the
acid reaction into alkali and will flourish the blue color).
 Place in eosin for 1-5 minutes.
 Contact time depends upon the efficacy of stain. Dip 1-2 times in water.
 It will give pink color to cytoplasm. Eosin is an acidic dye.

PAS (periodic acid & schiff’s) staining

 Firstly fix the slides with ethanol for 1 minute. Than dip the slides in runing water for 1-2
minutes.
 After this add periodic acid on slide for 5 minutes. Than place slide in runing water again
for 5 minutes.
 Add schiff’s reagent stain on the slides for 15 minutes. Again dip the slides rack in runing
water for 3-5 minutes.
 Place in hematoxylin stain for 1 minute.
 Give a quick dip to slides in water, than clear and mount the slides for observation as
shown in figure 2.23.

33
 A B

Figure 2.21 staining of racks holding slides of tissue showing

A=staining of slides in racks; B=stained slides

Mounting
 Stained slides are then dried to proceed for mounting.
 Mounting is the last step which is performed by using mounting reagent which is DPX.
 Place a drop of DPX on stained slide and cover it with cover slip in such a way that the
stained area may not remained dry.
 Leave it for drying. After it slides are examined microscopically.

34
Chapter-3: CONCLUSION & LIMITATION
3.1 Conclusion
This internship has provided an excellent and rewarding experience. Accurate and rapid diagnosis
procedures are provided through clinical microbiology. At Citi Lab & Research Centre, the
opportunity was afforded to work in the special chemistry department, where manual ELISA tests
were observed and hands-on practice was conducted. Additionally, practice was gained on
automation machines, including Abbott Alinity i and Roche by Hitachi Cobas 6000 + E411. In the
microbiology department, hands-on experience was gained in various techniques and procedures
for isolating and identifying microorganisms from patient samples and environmental sources
using microscopy, staining, and biochemical tests. Microorganisms were cultivated in controlled
environments, and their growth and characteristics were observed. Antimicrobial susceptibility
testing (AST) was conducted to determine the effectiveness of antibiotics against specific
microorganisms. In the histopathology department, hands-on experience was gained in processing,
examining, and diagnosing tissue samples. Tissue samples were grossed, sectioned, and stained,
and assistance was provided in microtomy.

3.2 Limitation of training program

Three months training was not enough time to fully engage with all aspects of clinical work or see
cases through completion. The limited duration of the internship may have restricted the depth of
experiences and learning opportunities.

35
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