CBME Microbiology Practical Record Apurba Sastry

GSL MEDICAL COLLEGE
DEPARTMENT OF MICROBIOLOGY
PRACTICAL RECORD
BOOK
For MBBS Phase II
A Model, based on National Medical
Commission India’s
Competency Based Medical Education (CBME) Curriculum
Fill the particulars given below

GSL MEDICAL COLLEGE
DEPARTMENT OF MICROBIOLOGY
Certificate
It is certified that, this is a bonafide record of the practical exercises of
Microbiology done byMiss / Mr your name
University. Reg. No. ___________________ He/She has attended all the exercises
entered in this record book and his/her progress is satisfactory.
Signature of the Teacher in-charge Professor and Head
Name: Department of Microbiology
Date:
Instructions to Students
1. Be punctual and maintain time specified for each exercise/activity
2. Eating or drinking in the lab at any time is to be strictly avoided
3. Report all accidents, injuries, and breakage of glass or equipment to instructor
immediately.
4. Keep pathways clear by placing extra items (books, bags, etc.) on the shelves or under the
work tables
5. Long hair must be tied back.
6. Do not enter lab without Lab coat. Loose clothing should be secured so that they do not
get caught in a flame or chemicals.
7. Come prepared with prior reading on the assigned experiments before you start to work.
8. Pay close attention to anycautions described in the laboratory exercises
9. Do not taste or smell chemicals.
10. Unauthorized experiments or procedures must not be attempted.
11. Keep solids out of the sink.
12. Do not lean, hang over or sit on the laboratory tables.
13. Do not leave your assigned laboratory station without permission of the teacher.
14. Learn the location of the fire extinguisher, eye wash station, first aid kit.
15. Do not lift any solutions, glassware or other types of apparatus above eye level.
16. Learn how to handlethe laboratory materials and equipment safely.
17. Follow all instructions given by your teacher.
18. Leave your work station clean and in good order after your work
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Table of Contents
S.No. Topic Competency Page Score Faculty
(*Certification Skills **AETCOM) No. No. Signature
I General Microbiology, Immunology & Hospital Infection Control
1 A-Introduction to Microbiology laboratory MI 1.1 8 - 13
B - Microscopy MI 1.2
2 1. General principles of Laboratory diagnosis of MI 8.9, 14 - 22
Bacterial diseases.(Sample collection, transportation, MI 8.10
bacterial identification methods in general) MI 8.7
2. Demonstration of respect for patient samples sent MI 8.11
for laboratory investigations**
3. Hand hygiene, PPE- Donning & Doffing of hand MI8.7 23 - 26
gloves (1)*
3 1. Direct methods of bacterial detection MI 1.2 27–28
a) Simple staining
b) Hanging drop
2. Gram staining (1)* MI1.2 29-31
4 Ziehl-Neelsen staining (1)* MI 1.2 32-35
5 Bacterial culture: Culture media, methods and MI 1.1 36-43
identification techniques (conventional and automated)
6 Antibiotic sensitivity testing MI 1.6 44-47
7 Indirect methods of infectious disease diagnosis MI 1.8, MI 48-52
(Immunological diagnostic tests) 8.15
8 General principles of laboratory diagnosis of Viral MI 1.1 53-55
diseases
9 1. General principles of laboratory diagnosis of MI 1.2 56-59
Parasitic diseases
2. Stool Examination (1)* MI1.2 60-61
10 General principles of laboratory diagnosis of Fungal MI 1.1 62-65
diseases
11 Hospital infection control – I: Hand hygiene, PPE MI8.5, MI8.6, 66-69
(Donning & Doffing) MI8.7, MI8.8
Hand hygiene, PPE (2)* MI8.7 70-73
12 Hospital infection control - II: Sterilization & MI8.5, MI8.6, 74-79
Disinfection, Biomedical waste management, Needle MI8.7, MI8.8
stick injuries
4
II Bloodstream and cardiovascular system infections
13 Laboratory diagnosis of Rheumatic heart disease, MI2.1, MI2.2, 80-85
Infective endocarditis and sepsis MI2.3
Gram Stain (2)* MI1.2 86-87
14 Laboratory diagnosis of Brucellosis, Leptospirosis, MI4.3, MI8.1 88-94
Dengue fever, Scrub typhus, Candidemia
15 Laboratory diagnosis of Enteric fever MI3.3, MI3.4 95-97
16 Laboratory diagnosis of Malaria MI2.5, MI2.6 98-101
17 Laboratory diagnosis of Filariasis and Leishmaniasis MI2.5, MI2.6 102-
105
18 1. Laboratory diagnosis of HIV infection MI2.7 106-
2. Confidentiality pertaining to patient's identity in MI8.12, 111
laboratory result** MI8.14
III Gastrointestinal &Hepatobiliary infections
19 Laboratory diagnosis of Diarrhea MI1.2, MI3.1, 114-
MI3.2 121
Ziehl-Neelsen staining (2)* MI1.2 122
Stool Examination (2)* MI1.2 124
20 Laboratory diagnosis of Dysentery MI1.2, MI3.1, 126-
MI3.2 128
Stool Examination (3) 129-
131
21 Laboratory Diagnosis of Intestinal helminthic MI1.2, MI2.4, 132-
infections MI2.5, MI3.1, 140
MI3.2
Stool Examination (4)* MI1.2 141
22 Laboratory diagnosis of Hepatic infections MI3.7, MI3.8 143-
151
IV Skin, soft tissue and musculoskeletal system infections
23 Laboratory diagnosis of skin infections-I MI4.3 154-
(Bacterial: Furuncle, cellulitis, Surgical site infection, 166
Burn wound infection, Leprosy)
Gram Stain (3)* MI1.2 155
Ziehl-Neelsen staining (3)* MI1.2 165
24 Laboratory diagnosis of skin infections-II MI4.3 167-
(Fungal and Viral infections) 175
25 Laboratory diagnosis of musculoskeletal infections MI4.2 176-
5
(Arthritis, Osteomyelitis) MI1.2 182
Ziehl-Neelsen staining (4)*
V Central nervous system infections
26 Laboratory diagnosis of Meningitis (Pyogenic, MI5.3 183-
Tubercular, Cryptococcal& Aseptic) 190
* Gram Stain (4) MI1.2 187
27 Laboratory diagnosis of Encephalitis MI5.3 189-
190
VI Respiratory tract infections
28 Laboratory diagnosis of Upper respiratory tract MI6.2 194-
infections 197
Gram Stain(5)* MI1.2, MI6.2 196
29 Laboratory diagnosis of Lower respiratory tract MI6.2, MI6.3 198-
infections 209
Ziehl-Neelsen stain (5)* MI1.2, MI6.3 200
Hand hygiene, PPE (3)* MI8.7 204-
207
VII Genitourinary & Sexually transmitted infections
30 Laboratory diagnosis of Urinary tract infections MI7.1, MI7.2 210-
214
31 Laboratory diagnosis of Genitourinary and Sexually MI7.3 215-
transmitted diseases (Urethritis, Genital ulcers) 217
6
BLOCK I
General Microbiology, Immunology &

Hospital Infection Control
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1A.Introduction to Microbiology Laboratory
Competencies
MI1.1: Describe the different causative agents of Infectious diseases and the methods used in their
detection, and discuss the role of microbes in health and disease
MI 1.2: Perform and identify the different causative agents of Infectious diseases by Gram Stain, ZN stain
and stool routine microscopy
Specific Learning Objectives: At the end the session, the students shall be able to,
 Describe the structure and functions of Microbiology laboratory

 Discuss the general principles and steps involved in the laboratory diagnosis of infectious diseases
(Emphasize on Koch’s postulates)
 Explain the Good laboratory practices
 Demonstrate practice of standard precautions
 Explain the concept of Biosafety in the laboratory
Exercise 1.A:
1. Enumerate various sections/divisions of Microbiology laboratory: describe their functions and major
diagnostic tests carried out in each section.
Section Diagnostic tests done
Bacteriology Staining & microscopy, culture, biochemical tests, serology tests,

molecular tests, antimicrobial susceptibility test. They help to identify
the bacterial pathogen in clinical specimens and its antibiotic sensitivity
pattern.
Virology Electron microscopy, cell culture, serology tests, molecular tests. They
help to identify the viral pathogen in clinical specimens.
Parasitology Microscopy, serology tests. They help to identify the parasitic pathogen
in clinical specimens.
Immunology Rapid immunochromatography tetsts, ELISA, FIA, CLIA, western blot

test, precipitation reactions, agglutination reactions. They are useful in
diagnosis of the infections.
Mycology Staining & microscopy, culture, biochemical tests. They help to

identify fungal pathogen in clinical specimens.
Molecular biology PCR, real time PCR, reverse transcriptase PCR, multiplex PCR. They
help in diagnosis of the infections.
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2. Describe laboratory method utilized for demonstration of Koch’s postulates for establishing the causative role of a pathogen in a disease
process with suitable example.
Koch’s Postulate Demonstration
1. Microorganism should be constantly Acid fast bacilli seen in tubercle lesions produced by
associated with the lesions of the Mycobacterium tuberculosis
disease.
2. It should be possible to isolate the M.tuberculosis is grown in pure culture from the lesions of the
organism in pure culture from the disease on Lowenstein Jensen medium
lesions of the disease.
3. Same disease must result when the TB lesions seen in rabbits, mice, guinea pigs.
isolated organism is inoculated into a
suitable laboratory animal.
4. It should be possible to re-isolate the M.tuberculosis is grown in pure culture from the lesions of the
organism in pure culture from the disease on Lowenstein Jensen medium
lesions produced in the animal.
5. Specific antibodies to the disease Variable antibody response seen

causative organism should be found
in patient serum.
3. Discuss the significance of proper hand hygiene
Hands of health care workers are the main source of infections to the patients. Proper hand hygiene is therefore
important to prevent healthcare associated infections. It is important to perform hand disinfection before donning sterile
gloves, before surgery and in between surgeries to prevent surgical site infections. It is important before performing
wound dressing to prevent wound infection. It prevents infections transmitted by contaminated hands and dropletes.
4. Enumerate the diseases that can be transmitted through contaminated hands
Staphylococcus aureus skin infections, Coagulase negative staphylococcal skin infections, Salmonellosis, influenza,
cold, COVID 19.
5. Suggest the appropriate type of biosafety cabinets to be used in the following scenarios
Scenario Biosafety level
BSC class I 1- provides Protection to the environment and the laboratory

personnel
BSC class II 2 - provides protection additionally to samples handled inside the

cabinet
BSC class III 3,4 - prevents direct contact between the operator and the samples
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1B. Microscopy
Specific Learning Objectives: At the end the session, the students shall be able to,
 Enumerate different type of microscopes
 Describe the working principle of compound microscope, Dark-field, Phase contrast,
Fluorescent and Electron microscopes
 Discuss the applications of various microscopes in diagnostic microbiology
 Focus the compound microscope under various magnification powers
 Describe the concept of Micrometry
 Handle the microscope with utmost care
Exercise1.B:
1) Enumerate different types of microscopes used in diagnostic microbiology laboratory

Light microscope. Dark field microscope. Phase contrast microscope. Fluorescence
microscope. Electron microscope.
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
2) Describe the principle and uses of various microscopes
a) Dark-field microscope
Principle: A special dark field condenser is used. It allows light to pass through and focus on the
specimen obliquely. Therefore only the light that is reflected by the specimen enters into the
objective lens which makes the object to appear bright against a dark background.
Uses: it is used to identify the living, unstained cells, and thin bacteria like spirochetes.
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11
b) Phase contrast microscope
Principle: It increases contrast. It visualizes the unstained living cells by creating difference in contrast between the
cells and water.
Uses: It is useful for studying microbial mobility, determining the shape of living cells, determining the microbial
internal cellular components. It converts slight differences in refractive index and cell density into easily detectable
variations in light intensity. The condenser produces a cone of light. The light rays pass through condenser,
specimen, phase ring in objective lens and then into ocular lens respectively. Finally, the background is bright,
while the unstained object appears dark and well defined.
c) Fluorescent microscope
Principle: It uses a fluorescence property to generate an image. Fluorescent dyes are exposed to UV rays and when
they become excited they fluoresce into visible light. The source of light is a mercury lamp which emits rays that
pass through an excitation filter.
Uses: It helps in visualizing microbes. Some microbes are self fluorescent. Some microbes fluoresce when they are
stained with specific fluorochrome dyes. Auramine phenol dye is used to visualize tubercle bacilli. The dyes can be
tagged to specific antibodies to detect specific antigens which helps in detection of microbes which is called as
immunofluorescence assay.
d) Electron microscope
Principle: It uses accelerated electrons as a source of illumination. It gives best resolution of 0.5 nanometers.
Electrons are generated by electron gun and they travel in vacuum through a magnetic condenser and then they
bombard on specimen. Specimen scatters electrons and then the electron beam is focused by magnetic lenses to
form an enlarged, visible image of the specimen on a fluorescent screen.
Uses: It is used in detection of viruses. It helps reveal details of flagella, fimbriae and intracellular structures of a
cell. Scanning electron microscope helps to examine the surfaces of microorganisms in great detail.
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3) Label the parts of microscope in the given picture
write parts as detailed in text book page No.6
4) Suggest the type of microscope, desired magnification and method used in the following case
scenarios
Case scenario Microscope Magnification Method
Stool examination for Light 400x Saline mount

parasitic eggs microscope
Urine examination for pus Light 400x Direct mount

cells in a case of Urinary tract microscope
infection
Bacterial examination of CSF Light 1000x Gram stain

sample in case of pyogenic microscope
meningitis
Bacterial examination of Dark field - -

tissue fluid from gummatous microscope
ulcer in a case of sexually
transmitted disease
5) Write the differences between Electron microscope and the Light/Compound microscope
13
Features Light/Compound microscope Electron microscope
Best resolution 0.2 microns 0.5 nanometers
Radiation source Visible light Electron beam
Medium of travel Air Vacuum
Specimen mount Glass slide Copper grid
Type of lens Glass Electromagnet
Highest practical 1000 – 1500 100,000

magnification
Assessment
Sl.No. Student’s performance Score#
1 Comes prepared with requisite prior knowledge 1 2 3

2 Participates actively and contributes to discussion during SGT 1 2 3
3 Shows professional conduct during the Teaching Learning 1 2 3
session
4 Completes the record book activities in time 1 2 3
5 Shows evidence of learning the new skills 1 2 3
(Intellectual/Psychomotor)
Total score /15
(Can be reduced to 5 for convenience)
Faculty Remarks/Feedback:
Date : Faculty Name & Signature
# Mark as 1, 2, 3 for ‘Not satisfactory’, ‘satisfactory’ & ‘Very Good’ respectively
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2. General Principles of Laboratory
Diagnosis of Bacterial diseases
Competencies
MI 8.7: Demonstrate Infection control practices and use of Personal Protective Equipment (PPE)
MI 8.9: Discuss the appropriate method of collection of samples in the performance of laboratory
tests in the detection of microbial agents causing infectious diseases
MI 8.10: Demonstrate the appropriate method of collection of samples in the performance of
laboratory tests in the detection of microbial agents causing Infectious diseases
MI 8.11: Demonstrate respect for patient samples sent to the laboratory for performance of
Specific Learning Objectives:
At the end the session, the students shall be able to,

 Discuss the need for role of microbiological investigations in diagnosis of infectious
diseases
 Explain the steps in laboratory diagnosis of infectious diseases/syndromes
 Select appropriate clinical specimen in the given case under investigation
 Collect the clinical specimen appropriately
 Identify the essential components of a ‘Sample request form’
 Discuss the significance of prompt transport of a clinical specimen to the laboratory
 Describe the general principles of bacterial culture and identification methods
 Demonstrate respect for patients’ samples received at the laboratory for testing.
15
Exercise 2.1:
1) Suggest suitable clinical samples to be collected in the following infections/diseases
Type of infection/disease Suitable Clinical sample to be collected
Conjunctivitis Conjunctival swabs

keratitis Corneal scrapings
endopthalmitis Aqueous or vitreous fluid
Otitis media Middle ear aspirate
Oral and dental infections Swabs, aspirates, tissue specimens
Meningitis CSF
Skin and soft tissue infections Aerobic – pus, exudates, swabs, aspirate from abscess,
tissue bits
Anaerobic – tissue specimens, aspirates
Upper respiratory tract infections (Pharyngitis, Nasal swab, throat swab, nasopharyngeal swab
tonsillitis, diphtheria)
Lower respiratory tract infections (Bronchitis, Sputum, endotracheal aspirate, BAL, lung biopsy
Pneumonia, pulmonary tuberculosis)
Septicemia Pair of 8 – 10 ml blood samples from different sites

Endocarditis
Diarrhea, Dysentery, Food poisoning Stool, rectal swab, food sample
Genital infections (Gonorrhea, syphilis, chancroid Swabs from urethra, cervix. Exudates from ulcers
etc.)
Urinary tract infections Urine – MSU, suprapubic aspirate, from catheter,
Diseases requiring serological/immunological 2ml Blood in vacutainer

tests
2) Describe the precautions to be taken while collecting samples for anaerobic culture.
_1. Hand hygiene 2. Appropriate PPE 3. Aseptic measures. 4. Disinfection of patient care items. 5. Sample
collected before starting antibiotics. 6. Avoid contamination 7. Aspirates, blood, body fluids, tissue
specimens preferable depending on the site of infection. 8. Proper labeling. 9. Sample collected in Suitable
anaerobic culture media. 10. Sample should not be exposed to oxygen. Enumerate the culture media and
methods used for anaerobic culture.
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3. Enumerate the culture media and methods used for anaerobic culture ?
Anaerobic culture media Anaerobic culture methods
Robertson’s cooked meat broth 1. Evacuation and replacement

Thioglycollate broth 2. Oxygen absorption by chemicals by
Anaerobic blood agar gas pak system using anaerobic jar
BHIS agar with vit K and hemin 3. Anaerobic glove box and anaerobic
Neomycin blood agar work station
Egg yolk agar
Phenyl ethyl agar
Bacteroides bile esculin agar
4. What are the criteria for sample rejection?

1. Improperly labeled or unlabeled specimen_2. Incomplete information on request form
3. Insufficient specimen 4. Inappropriate specimen 5. Sample delayed in transit more than
accepted limit.
5.What are the general principles to be followed for specimen collection?
_1. Hand hygiene 2. Appropriate PPE 3. Aseptic measures. 4. Disinfection of patient care
items. 5. Sample collected before starting antibiotics. 6. Avoid contamination 7.
Specimen collected in sterile, wide mouthed, screw capped, tightly sealed, leak proof
containers. 8. Proper labeling. 9. Specimen for anaerobic processing should not be
exposed to oxygen and collected in anaerobic media. 10. Properly filled test requisition
form. 11. Specimen should not be sent in formalin. 12. Specimen collected depending on
the site of infection and the test requested.
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6. Label the blank parts of the packed clinical specimen in the following picture
See page No.225 in this record and label
(Source: Center for Disease Control, Atlanta, USA)
7. Write the general guidelines for specimen transportation

Specimen Transport time to the laboratory generally should not exceed 2 hours. But
Specimens such as CSF, body fluids, tissue specimens, bone, suprapubic aspirate, ocular
specimens should be transported to laboratory immediately within 15 minutes. If
required, appropriate transport media should be used. Specimen should not be transported
in formalin.
8. What are the important and essential components of specimen label?
Patient Name, age, gender, name of the treating physician, clinical diagnosis, antibiotic
history, type of specimen, desired investigation name.
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9. Following is the sample of specimen request form. Please fill the details as per the
clinical case discussion in this practical exercise.
19
10. Enumerate the steps used in bacterial culture
1. Selection of media.
2. Inoculation of the specimens
3. Incubation at 37 degrees Celsius in a incubator for 24 to 72 hours usually
11. Write the methods used for bacterial identification
1. Colony morphology properties

2. Gram stain of Culture smear
3. Motility test by hanging drop examination
4. Biochemical reactions
5. Antimicrobial susceptibility test
6. Serodiagnosis
7. Molecular methods
8. Typing
12. Write in brief on professional conduct pertaining to the clinical samples
Essential background knowledge required is :

1. Appropriate sample for the test planned: sample type, amount, collection
procedure, preservative if any, container type used, its transportation and storage.
2. Appropriate labeling for correct sample identification
3. Accompanying clinical information for correlation
4. Possible medicolegal, ethical and sociocultural issues following
incomplete/incorrect sample identification, improper storage or transportation
5. To apply rejection criteria
6. To prioritize sample processing
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AETCOM Exercise
Exercise 2.2: Demonstrate respect for patient samples
Competency:
MI 8.11 Demonstrate respect for patient samples sent to the laboratory for performance of

At the end of the module students should be able to
 Describe and apply professional and moral values related to patients’ clinical samples at
the sample reception and processing stage
 Identify the routine events in sample collection, transportation and storage that are
disrespectful to clinical sample.
 Identify the ways to rectify them and demonstrate respect to patients’ samples.
 Describe and demonstrate application of ethical principles and medico legal issues in
laboratory results
In the given situation/case scenario, identify the events that show disrespect / breach in
confidentiality to the clinical sample/ data and suggest ways to rectify them.
____________________________________________________________________________
1) Biopsy sample of a critical patient getting rejected because it was collected in a formalin
saline
It should be mentioned that biopsy is received in formalin, hence unsuitable for culture.
There is no more specimen available for culture now. This is done in the best interest of the
patient care.
Specimen should not be sent in container containing formalin for microbiological analysis.
Therefore biopsy is rejected. The information should be made well aware in the hospital.
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2) Left over serum sample getting used for research without informed consent
Sample using for research without taking prior consent from the patient is a breach in
confidentiality to the clinical sample.
Patient is privileged and it is his right to be well informed of what is going to be done with his
sample. Hence, it is our responsibility to make patient well aware of the research done with his
sample. Informed consent should be taken from the patient before subjecting his sample to
research work. Patient should be informed about the consequences of the results obtained in the
research work and assurance given pertaining to confidentiality.
3) Sample is accompanied with request form with incomplete information/ wrong patient
credentials
Specimen should be taken for testing only with a request form containing complete information
of the patient. If the patient information is incomplete or wrong, the sample is rejected and the
concerned clinical team is informed about the information that should be written on the request
form and sample container, completely and legibly. Usually the information will be patient
name, gender, age, ward, hospital number, sample type, clinical diagnosis, treatment history.
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4) Undue delay in testing and reporting of a sample requiring emergency testing
This situation tells the importance of prioritizing the specimen as relevant to the
clinical case. The laboratory should be aware of prioritizing a sample requiring
immediate processing and reporting over the others. Certain precious samples like
CSF, sterile body fluids, ocular specimens, tissue specimens, supra pubic aspirate,
bone specimen should be processed immediately as soon as received, not more
than 15 min delay. Similarly, blood culture bottles should be immediately
incubated upon receipt.
Assessment

3 Shows professional conduct during the Teaching Learning session 1 2 3
Total score /15
23
Certifiable Skill Exercise
Exercise 2.3: Hand hygiene, PPE - Donning & Doffingof hand gloves – (1)
1) Perform hand hygiene appropriately and in the correct sequence
(Source: Essentials of Practical Microbiology, 2nd edition)
2) Write examples of situations requiring hand hygiene in an outpatient department

1.Before touching a patient. 2. Before clean or aseptic procedures. 3. After body fluid
exposure. 4. After touching a patient. 5. After touching patient’s surroundings.
3) Perform donning and doffing of gloves appropriately
Steps of donning (wearing) gloves – left picture shows donning first glove. Wear by
touching and pulling only the edge of the cuff.
Right picture shows donning second glove. Avoid touching the forearm skin by
pulling external surface of second glove by the finger of gloved hand.
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Steps of doffing (removal) gloves – do not touch the outside of the gloves as they are
contaminated. Using the gloved hand, grasp the palm area of the other gloved hand
and peel off first glove. Hold the removed glove in gloved hand. Slide fingers of the
ungloved hand under the other glove at wrist and peel off second glove over the first
glove. First glove will remain inside the pouch of the second glove. Perform hand
hygiene after removal.
4) Write examples of situations requiring use of gloves in an out-patient department

1. Before doing a sterile procedure. 2. During contact with non intact skin and mucus
membranes. 3. When contact with blood or body fluids is anticipated. 4. During
contact with the patient and his immediate surroundings. 5. Heavy duty gloves used
to protect from sharp injuries.
5) You are about to perform phlebotomy for serological testing. Choose appropriate PPE for the
procedure. Demonstrate the appropriate method of donning and doffing of the selected PPE.
Perform hand hygiene where ever
PPE needed are a pair of gloves, gown, surgical mask, protective eyewear. Hand hygiene
performed before and after the phlebotomy procedure. Technique is as explained above.
25
Checklist for Assessment of Certification Skill.
Hand hygiene
Step Student’s performance Score YES NO

(Score=0)
1 Removes all hand accessories( finger ringwrist watch. etc.) 1.0
(0.5) and
Applies sufficient amount of soap/hand wash /hand rub (0.5)
2 Rubs palm to palm 0.5
3 Rubs back of palm on Both sides (0.5 + 0.5) 1.0
4 Follows rotational rubbing of thumb on Both sides (0.5 + 0.5) 1.0
5 Rubs back of fingers on palm on Both sides (0.5 + 0.5) 1.0
6 Interlaces fingers in the web spaces 1.0
7 Rubs nails on palms on Both sides (0.5 + 0.5) 1.0
Completes the above steps in 20-40 seconds time or 1.0
Waits till the hands are dried (in case of hand rub)
8 Rinses hands with water 0.5
9 Dries hands with paper with single use towel & (0.5) 1.0
Closes the tap with same paper towel/elbow (0.5)
10 Disposes the paper towel appropriately 1.0
Completes the steps 8, 9 & 10 in 40-60 seconds time 1.0
Total score 10 /10
26
Donning (wearing) & Doffing (removal) of Hand Gloves

(Score=0)
Donning Glove:1 Wears by touching and pulling only the edge of 1.0
the cuff
Glove:2 a) Wears by pulling the external surface of 1.0
second glove by
the finger of gloved hand (0.5) and
b) avoids touching the forearm skin (0.5)
Doffing Glove:1 Removes first glove by using the other gloved 1.0
hand, grasps the palm area of the first glove &
peels it off.
Glove:2 a) Holds the removed glove in the other gloved 1.0
hand (0.5) and
b) slides fingers of the ungloved hand under the
other glove at wrist and peels off second glove
over first glove. (0.5)
a) Disposes the gloves appropriately(0.5) and 1.0
b) Performs hand hygiene after gloves removal (0.5)
Total score 5.0 /05
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3. Direct methods of bacterial detection
Competency
MI 1.2: Perform and identify the different causative agents of Infectious diseases by Gram Stain

 Enumerate various types of staining techniques used in bacterial identification
 Describe the principles of simple staining, hanging drop.
 Identify the different causative agents of Infectious diseases by simple staining &
hanging drop.
 Describe the principle of Gram staining
 Perform Gram stain on the smear provided, record the observations with help of a
colored labelled diagram and interpret the findings.
 Identify different causative agents of infectious diseases by Gram Stain
Exercise 3:
1) Record the findings of simple staining :
Fig.3.2.1
Draw clusters in blue color Draw bacilli in pink color
Methylene blue stained smear shows Basic fuchsin stained smear shows
Blue colored cocci in clusters pink colored bacilli in scattered
arrangement
2) Write the clinical applications of simple staining
Bacteria cannot be seen under light microscope due to lack of contrast. Bacterial cytoplasm is
acidic in nature. They take the color when stained with basic dyes such as methylene blue or
basic fuchsin and therefore appear with color contrast under microscope. Simple stain imparts
same color to all the bacteria in the smear. It is useful to tell quickly whether the sample contains
bacteria or absent. Bacterial size, shape, arrangement can also be observed.
28
3) Record the findings of Hanging drop
Edge of the drop under 10x Edge of the drop under 40x
Showing motile bacilli
4) Write the clinical applications of Hanging drop
It is useful to demonstrate the bacterial motility. Bacteria moving in different directions and with
changing their positions in the field are said to be actively motile.
Assessment

Total score /15
29
# Mark as 1, 2, 3 for ‘Not Certifiable Skill Exercise satisfactory’, ‘satisfactory’ & ‘Very
Good’ respectively
Gram Stain
1) Write the principle of Gram stain
Gram stain differentiates bacteria into gram positive bacteria and gram negative bacteria.
Bacteria that appear violet after Gram stain are said to be gram positive bacteria. Bacteria
that appear pink after gram stain are said to be Gram negative bacteria.
Gram positive bacteria retain primary stain during decolorisation because of their strongly
acidic cytoplasm which holds the dye more firmly and thick, impermeable cell wall which
acts as a barrier and prevents loss of the dye during decolorisation. Also alcohol decoloriser
shrinks cell wall pores.
Gram negative bacteria lose primary stain during decolorisation because of their weekly
acidic cytoplasm which could not hold the dye firmly and thin cell wall which becomes
permeable to the dye during decolorisation. Also decoloriser disrupts LPS in cell wall
which makes cell wall pores larger in size.
2) Perform Gram staining, focus under the microscope, record the observations and interpret
the result of Gram staining :
Fig.3.3.2A
Observation 1. Violet colored cocci in clusters

2. pink colored bacilli in scattered arrangement seen
Inference 1. Gram positive cocci 2. Gram negative bacilli
Examples 1. Staphylococci 2. Escherichia coli
30
Gram positive cocci in clusters Gram positive cocci in chains
Fig.51.2B Fig.52.4B
Gram positive cocci in pairs Gram positive bacilli in chains
Fig.61.1 Fig.55.2A
Gram negative bacilli Gram negative cocci in pairs
Fig.41.1B Fig.71.2
3) What is the crucial step in the Gram staining and why?

Decolorisation is the most crucial step in Gram stain procedure. If it is applied for more
time, even Gram positive bacteria gets decolorized and hence take pink stain and appear
pink giving the impression of Gram negative reation. Similarly, if it is applied for less
time, even Gram negative bacteria do not lose their primary stain and appear violet giving
the impression of gram positive reaction.
31
4) List the differences between Gram positive and Gram negative cell walls
Gram positive cell walls Gram negative cell walls
Peptidoglycon layer - thick Thin
Teichoic acid - present Absent
Lipopolysaccharide – absent Present
Lipid content, amino acids – nil/scanty Present
5) Write the clinical applications of Gram stain

To differentiate bacteria into gram positive and gram negative group is useful in
identification of the bacteria. Bacterial size, shape, arrangement also helps to identify the
bacteria and helps physician in determining the treatment empirically.
Skill certification
Competency No.: MI 1.2 Competency: Gram Stain
Student’s Performance Max. Marks Marks
(05) Scored
Performs skill by following all the steps correctly 02
Focusses the stained slide appropriately 01
Identifies the structures correctly and interprets. 01 (0.5+0.5)
Draws colored labelled diagram of the microscopic field and 01 (0.5+0.5)

writes the report
Score
Rating Rubric Rating

Below expectations (B) (Score – 1, 2, 2.5)
Meets expectations (M) (Score – 3, 3.5)*
Exceeds expectation (E) (Score – 4, 4.5, 5)
32
CERTIFICATION NO YES
(*Students should secure ‘M’ or ‘E’ to be able to get

Certification in a given skill)
Date Faculty Name & Signature
33
4.Ziehl-Neelsen staining
Competency
MI 1.2: Perform and identify the different causative agents of Infectious diseases by ZN stain
 Describe the principles of Ziehl-Neelsen stain.
 Perform Ziehl-Neelsen stain on the smear provided, record the observations by drawing
neat labeled diagram with appropriate colors and interpret the findings.
 Identify different acid fast organisms/structures causing infectious diseases by Ziehl-
Neelsen Stain
 Describe and apply RNTCP grading for suggesting therapy.
Exercise 4:
1) Perform Ziehl-Neelsen staining on the given heat-fixed sputum smear from a 42-year-old
lady with cough with expectoration, evening rise of temperature and loss of weight since 1
month. Focus the smear under microscope, record your observations and interpret the results
Fig.63.3A
Observations Long, slender, some are straight, some are slightly curved, some are beaded,
red colored bacilli seen among blue colored pus cells and epithelial cells.
Inference Acid fast baciili.
Examples Mycobacterium tuberculosis
34
2) Describe the principle of Ziehl-Neelsen stain
It consists of 4 steps :
1. 1% strong carbol fuchsin [ primary stain ] is applied on sputum smear for 5 minutes.
Intermittent heating done under the smear until vapor rises. Wash with water.
2. Apply 25% concentrated sulfuric acid on smear for 2 – 4 minutes. Wash with water.
3. Clean the surface of the slide back to the smear. Then 0.1% methylene blue is applied for
30 seconds. Wash with water. Dry the smear with tissue paper.
4. Examine under oil immersion objective lens. Then discard the slide and tissue paper in a
jar with 5% phenol.
3) Enlist various acid fast organisms/structures and the percentage (%) of decolorizer (sulfuric
acid) used for each type.
Acid fast organisms/structures Percentage (%) of decolorizer
M.tuberculosis 25
M.leprae 5
Nocardia 1
4) What is Kinyoun’s method ? Write its applications.
It is a modification of acid fast stain. In this method heat is not applied. 0.5% sulfuric Acid
is used as decoloriser. Malachite green is used as secondary stain. It is used to stain parasites
such as cryptosporidium, cystoisospora, cyclospora.
5) Write the principle of Ziehl-Neelsen’s stain

Acid fast bacilli show resistance to decolorisation by strong acids. This is due to the presence
of mycolic acid in their cell wall which is a candle wax like fatty substance. It makes cell
wall impermeable to stain the bacteria directly. Therefore, to stain these bacteria, heat is
applied which liquifies the mycolic acid and therefore cell wall becomes permeable to the
primary red stain. Once, heat is withdrawn mycolic acid solidifies because of its wax like
nature and makes cell wall impermeable to stain. Therefore primary stain is not lost in
decolorisation and bacilli appear red. Non acid fast cells get decolorized because of the
absence of mycolic acid and therefore they take the secondary blue stain and appear blue.
6) Record the observations of Ziehl-Neelsen’s stain

35
Fig. 63.3A Fig.54.3
M.tuberculosis M.leprae
Nocardia Coccidian parasites
Fig.55.5 Fig.45.11A
7) Explain RNTCP grading

Grade 1 : no AFB found in 100 OIF – negative
Grade scanty : 1 – 9 AFB seen in 100 OIF – positive
Grade 1+ : 10 – 99 AFB seen in 100 OIF – positive
Grade 2+ : 1 – 10 AFB seen per OIF – positive
Grade 3+ : >10 AFB seen per OIF – positive
8) Describe the therapeutic implication of RNTCP grading
1. Useful to monitor response of the patients to treatment

2. Useful to assess the severity of the disease
3. Useful to assess the infectious nature of the patient
36
Skill Certification
Competency No.: MI 1.2 Competency: Ziehl-Neelsen Stain (1)
(05) Scored

writes the report
Score


37
5. Bacterial culture and identification
Competency
MI1.1: Describe the different causative agents of infectious diseases and the methods used in
their detection, and discuss the role of microbes in health and disease
Specific Learning Objectives:At the end the session, the students shall be able to,
 Explain the purpose of growing the bacteria in the laboratory
 Enlist the basic nutrients required in a culture medium for the growth of bacteria
 Enumerate various culture media used for growing bacteria
 Define different types of culture media (Enriched, Enrichment, Selective, Transport,
differential) and explain their uses
 Suggest the use of suitable culture media based on the type of clinical specimen and
condition
 Enumerate the media, methods and uses of anaerobic culture
 Describe the media, methods and uses of blood culture
 Discuss the principle of automated blood culture systems with examples
 Compare and contrast the conventional from automated blood culture method
 Enumerate the methods/tests used for presumptive identification of bacteria from culture
Exercise 5:
1) Explain the purpose of growing the bacteria in the laboratory
Bacterial culture is the most common diagnostic method used for detection of bacterial
infections. Specimens are inoculated on to various culture media and incubated. The bacterial
colonies grown are subjected to identification and antimicrobial susceptibility test.
2) Write the basic chemical constituents in a culture medium
Water, electrolytes like Nacl, peptone, agar, meat extract, yeast extract, blood and serum.
38
3) Enumerate different types of transport media and indicate the clinical conditions and type of bacteria
that can be supported by such media
Transport medium Clinical condition Bacteria
Aerobic:
1) Amies or Stuart’s Gonococcal urethritis Neisseria gonorrhoeae
2) Venkata Raman cholera Vibrio cholera
3) Cary Blair cholera Vibrio cholera
4) Buffered glycerol dysentery Shigella

saline
Anaerobic:
1) RCM broth OT sterilization Clostridium tetani
2) BHI broth Endocarditis Bacteroides fragilis
4) Define different types of culture media (Enriched, Enrichment, Selective, differential) and explain
their uses
Type of media Examples Uses
Enriched Blood agar Demonstrates hemolytic

media: property of bacteria
Chocolate agar Useful to grow Haemophilus

influenza
Loeffler’s serum slope Useful to grow

Corynebacterium diphtheria
Blood culture media Useful in isolating bacteria

from blood
Enrichment Tetrathionate broth Used for Salmonella typhi

media:
Gram negative broth Used for Shigella
Selenite F broth Used for Shigella
Alkaline peptone water Used for Vibrio cholera
39
Selective media: Lowenstein Jensen medium Used to isolate Mycobacterium
tuberculosis
Thiosulfate citrate bile salts sucrose agar Used to isolate Vibrio cholera
Deoxycholate citrate agr Used to isolate Shigella and

Salmonella
Potassium tellurite agar Used to isolate Corynebacterium

diphtheria
Differential MacConkey agar Differentiates lactose fermenting

media: bacterial colonies fron non lactose
fermenting bacterial colonies
CLED agar Differentiates LF from NLF

colonies isolated from urine
samples
5) Draw the colored labelled diagrams of culture media (without and with growth) in the space provided below
Blood agar with hemolytic colonies
Blood agar
Fig.3.3.4C Fig.3.3.16
MacConkey agar with

Lactose fermenting
Mac Conkey Fig.41.1A
colonies
agar
Fig.3.3.6C
Potassium tellurite agar

Chocolate AgarFig.
with black colored
Fig.3.3.4D fig. 60.4B
colonies
40
Fig.60.4A Loeffler’s serum slope
Fig.3.3.5A
Loeffler’s seru Lowenstein Jensen medium
TCBS agar Deoxycholate citrate

agar
Fig.3.3.5B Fig.3.3.6A
6) What is blood culture? Enlist the media, methods and uses of Blood culture
Recovery of bacteria from blood is called blood culture. As blood consists of inhibitory
substances and as many blood pathogens are fastidious in nature, enriched media are used for
isolating bacteria from blood. Monophasic and biphasic brain heart infusion media [ BHI broth
and BHI agar ] are commonly used media in conventional blood culture.. Blood culture is useful
in detecting the pathogen in bacteremia, enteric fever, brucellosis and septicemia conditions
7) Enumerate the automated blood culture systems. Add a note their principle
Automated blood culture methods are BacT/ALERT 3D, BacT/ALERT VIRTUO and
BACTEC. They monitor periodically for bacterial growth and once growth is detected
they give a signal in the form of a beep or color change on the screen. Tryptic soy broth
and BHI broth are commonly used.
41
8) Discuss the advantages and limitations of conventional and automated blood culture systems
Blood culture system Advantages Limitations
Conventional Low cost Cumbersome
Colony morphology can be Time consuming

observed
Automated Rapid High cost
Less labor intensive Colony morphology cannot be

observed
9) List the anaerobic culture media

Robertson’ s cooked meat broth, thioglycollate broth, anaerobic blood agar, BHI agar with
vitamin K and hemin, neomycin blood agar, egg yolk agar, phenyl ethyl agar, Bacteroides bile
esculin agar.
10) Describe the anaerobic culture methods and write their uses.
1. Evacuation and replacement method by using manual or automated anaerobic jar – now not in
use
2. Absorption of oxygen by chemical method using gas pak systemor GENbag – useful when
sample load is less. Commonly used method.
3. Anaerobic glove box and anaerobic work station - provides facility for easy sample
processing, incubation, examination of the specimens without exposure to oxygen.
4. Reducing agents like chopped beef heart particles are used in RCM broth
5. pre reduced anaerobically sterilized media.
11) Give examples for presumptive identification of bacteria based on colony characteristics.
Colony characters Probable bacteria
Bluish green colonies on nutrient agar Pseudomonas aeruginosa
Golden yellow colonies on nutrient agar Staphylococcus aureus
Large, mucoid pink colonies on MacConkey agar Klebsiella pneumoniae
Yellow colonies on TCBS agar Vibrio cholera
42
12) Enumerate the biochemical tests used for presumptive identification of bacteria.
Oxidase test Urea hydrolysis test Mehtyly red reaction test
Catalase test Triple sugar iron test Voges Proskauer test
Indole test Oxidation fermentation test Coagulase test
Citrate utilization test Sugar fermentation test Bile solubility test
13) Draw the colored labelled diagrams of key biochemical tests (Positive and Negative) in the
space provided below and write examples.
a) Catalase Test b) Oxidase test
Fig.3.3.17 Fig.3.3.18A
c) Indole test Fig.3.3.18B d) Citrate test Fig.3.3.19A
43
e) Urease test Fig.3.3.19B
f) Triple sugar iron agar Fig.3.3.20 A to F
14) What is MALDI-TOF? Discuss its clinical utility in diagnostic bacteriology.

It is Matrix assisted laser desorption/ionization time of flight. It identifies bacteria, fungi,
mycobacteria with a turnaround time of few minutes and with absolute accuracy. It
examines the pattern of ribosomal proteins present in the organism. The test isolate is
identified by comparing its spectrum with a known database.
44
Assement

session
Total score /15
45
6. Antibiotic sensitivity testing
Competency
MI1.6: Describe the mechanisms of drug resistance, and the methods of antimicrobial
susceptibility testing and monitoring of antimicrobial therapy
 Discuss the clinical significance of drug resistance in bacteria.
 Describe the methods of antimicrobial susceptibility testing in the laboratory
 Enumerate the antimicrobials to be used against Gram positive and Gram negative group
of bacteria
 Interpret the antimicrobial susceptibility test results and suggest therapy
Exercise6:
1) Explain the significance of antimicrobial susceptibility testing in the laboratory

AST guides clinician in choosing the right antibiotics in the management of infectious
diseases.
2) Describe the principle and utility of Kirby Bauer Disc diffusion test
It is the most widely used AST method. It is suitable for rapidly growing bacterial
pathogens. First, a lawn inoculum of the pathogen is made on Mueller-Hinton agar
medium. Then antibiotic disks known to be active against the pathogen are impregnated
on the surface of the medium. Then the agar plate is incubated at 37 degrees celcius for
18 hours. Zone of inhibition of bacterial growth around an antibiotic is suggestive of
sensitivity. Absence of zone of inhibition is suggestive of resistance. Zone diameter in
between sensitive zone and resistance zone is called as intermediate zone.
3) Draw a labeled diagram of Kirby Bauer Disc diffusion test with zones of inhibition indicating
sensitive (S), resistant (R) and intermediate (I) results.
Resistant (R) Sensitive (S)
Fig.3.3.23
Intermediate46
(I)
4) Write the recommended antimicrobials to be used against Gram positive and Gram negative
bacteria
Bacteria Antibiotics
Gram positive Penicillin Clindamycin
Vancomycin Cloxacillin
Gram negative Gentamicin Cefotaxime
Ciprofloxacin Imipenem
5) List the first, second line and the reserved antibiotics used against gram negative bacilli
Category Antibiotics
First line Gentamicin Amikacin
Ciprofloxacin Cefotaxime
Second line Amoxicillin+clavulanate Ampicillin+sulbactam
Levofloxacin Aztreonam
Reserved Imipenem Meropenem
Polymixin B Colistin
6) Explain what do sensitive (S), resistant (R) and intermediate (I) mean in the antimicrobial
susceptibility test reports?
Sensitive (S) Antibiotic is clinically effective when used in standard therapeutic

dose and can be used in treatment.
Resistant (R) Antibiotic is clinically not effective in standard dose or increased

dose and therefore should not be used in treatment.
Intermediate (I) Antibiotic is not clinically effective when used in standard dose but
may be effective when used in increased dose. They are not used in
treatment if alternate antibiotics are available.
47
7) Describe the principle and utility of Minimum Inhibitory Concentration technique (MIC) test.
The antibiotic is serially diluted and each dilution is tested with the testing pathogen for
antibiotic susceptibility. The lowest concentration of the antibiotic that will inhibit the visible
growth of the pathogen after incubation overnight is called MIC of the antibiotic for the tested
pathogen. MIC will guide clinician in selecting the most appropriate antibiotic and in planning
the dose of the antibiotic in the management of infectious diseases more accurately particularly
in critical illnesses like endocarditis, meningitis, pneumonia.
8) Describe the principle and utility of Epsilometer/E-test.

It is a quantitative method of estimating MIC. It uses an absorbent strip containing predefined
gradient of antibiotic concentration immobilized along its length. The strip is applied on to a
lawn inoculum of a testing bacterial pathogen. Following incubation, an elliptical zone of
inhibition is produced surrounding the strip. The antibiotic concentration at which the ellipse
edge intersects the strip, is taken as MIC value.
9) Draw a labeled diagram of Epsilometer/E-test
Fig.3.3.25
10) Enumerate the automated methods available for antimicrobial susceptibility testing
VITEK 2 identification and antimicrobial sensitivity system
Phoenix system
Micro scan walk away system
48
Assessment

session
Total score /15
49
7. Indirect methods of disease diagnosis
(Immunological tests)
Competencies
MI1.8: Describe the mechanisms of immunity and response of the host immune system to
infections
MI8.15: Choose and interpret the results of the laboratory tests used in diagnosis of the infectious
diseases
 Enumerate different immunological tests available for diagnosis of infectious diseases
 Describe the principle, types and application of Agglutination test
 Describe the principle, and application of Precipitation test
 Describe the principle, application of ELISA test
 Describe the principle, types and application of Immunofluorescence test
 Discuss the principle, Rapid Point of Care (POC) tests with examples.
Exercise 7:
1) List various types of immunological tests available for the diagnosis of infectious diseases
Conventional methods Newer methods
Precipitation ELISA
Agglutination CLIA
Complement fixation Western blot
Neutralization Immuno chromatography : flow through

and lateral flow assays
IFA
50
2) Describe the principle, types and applications of agglutination tests
Agglutination test principle:
When a particulate or insoluble antigen is mixed with its antibody in the presence of
electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated.
Types Examples Application
Direct agglutination Slide method Bacterial detection
Direct agglutination Tube method Antibody titer estimation
Indirect or passive Latex agglutination Antibody detection

agglutination
Hemagglutination Coombs test Rh antibody detection
3) Describe the principle, types and applications of precipitation tests
Precipitation test principle:
When a soluble antigen reacts with its antibody in the presence of optimal temperature,
pH and electrolytes such as Nacl, an antigen antibody complex is formed in the form of a
insoluble precipitate or floccules.
Flocculation Slide flocculation Diagnosis of syphilis
Precipitation Elek’s gel precipitation Detection of Toxin producing

Corynebacterium diphtheriae
51
4) Describe the principle, types and applications of ELISA
ELISA principle:
It is an immunoassay that detects either antigen or antibody in the specimen by using
enzyme+substrate+chromogen complex. An absorbing material is also used that
specifically absorbs the antigen or antibody present in serum. The enzyme is activated on
Antigen antibody combination and acts on substrate chromogen complex producing a
color change. The color change is detected by spectrophotometry in an ELISA reader.
Direct ELISA Detection of antigen HBsAg detection
Indirect ELISA Detection of antibody Detection of antibody in HIV
Sandwich ELISA Detection of antigen
IgM antibody capture Detection of antibody in dengue

ELISA
ELISPOT test Interferon gamma assay Detection of latent TB
5) Draw colored labeled diagrams of principle of Antigen and Antibody detection ELISA
reactions in the space provided below.
Fig.12.7 A Fig.12.6B
Sandwich ELISA Indirect ELISA
52
6) Describe the principle, types and applications of Immunofluorescence test
Immunofluorescence test principle:

A fluorescent dye conjugated with a specific antibody is used to detect specific antigens
on the cell surface. The dye absorbs high energy UV rays and in turn emit visible low
energy light.
Direct IFA Antigen detection Rabies antigen in corneal smear
Indirect IFA Antibody detection Antinuclear antibody in autoimmune

diseases
Flowcytometry Cell analysis CD4 cell count estimation
7) Draw colored labeled diagrams of Direct and Indirect Immunofluorescence (IF) reactions
below.
Fig.12.10A and B
Direct IF test
Indirect IF test
8) Discuss the principle of Rapid/ Point of Care (POC) tests with examples.
Rapid test principle:

They are simple to perform, takes only 10 – 20 minutes, require minimal training, do not
need any sophisticated instruments. They can be performed on field.
ICT - Lateral flow assay Cassette format HBsAg detection
ICT - Lateral flow assay Strip format HBs Ag detection
ICT - Flow through assay Cassette format HIV Ab detection
53
9) Following are the results of immuno-chromatographic test for Hepatitis B infection done on
three cases. Interpret your results and report (C=Control band, T=Test) Fig.12.12A and B
Case Result Report
A C T Negative
B C T Positive
C C T Invalid
Assessment

session
Total score /15
54
8. General principles of Laboratory
diagnosis of Viral diseases
Competency
MI1.1: Describe the different causative agents of Infectious diseases and the methods used in
 Discuss the purpose of laboratory diagnosis of viral infections
 Describe how the laboratory diagnosis of viral infections differ from bacterial disease
diagnosis
 Discuss the general steps in the diagnosis of viral infections
 Enumerate the direct and indirect methods for diagnosis of viral infections
 Describe the significance of viral inclusion bodies in disease diagnosis
 Describe Polymerase Chain Reaction and its role in laboratory diagnosis of viral
infections
Exercise 8:
1) Explain the need for laboratory diagnosis of viral infections
It plays an important role in establishing the specific diagnosis of various viral infections. It is
useful 1. to start specific antiviral drugs 2. to screen blood donors for HIV, HBV, HCV 3. For
surveillance purpose 4. For outbreak/epidemic investigation of viral infections like
influenza/dengue 5. To start post exposure prophylaxis of ART 6. To initiate TOP if rubella is
diagnosed in first trimester and to initiate HBIG in newborn diagnosed to have HBV infection.
2) Write the differences between laboratory diagnosis of viral infections and bacterial infections
Viral diagnostic methods Bacterial diagnostic methods
Electron microscope required to detect Light microscope is sufficient

viruses in clinical specimens
Cell lines required to culture viruses Artificial media are sufficient
Specific Cytopathic effects are diagnostic No specific CPE seen
55
3) Enumerate the direct and indirect methods for diagnosis of viral infections with examples
and their applications
Viral Examples Applications

diagnostics
Direct Electron microscopy Detection of rotavirus in stool sample

methods
Fluorescent microscopy Detection of rabies virus antigen in corneal
smear
Light microscopy Detection of virus specific inclusion bodies
Indirect ELISA Detection of specific viral antigens &

methods antibodies
Viral culture Isolation of virus by cell culture
PCR Detection of virus specific genes
4) What are inclusion bodies?

They are aggregates of virions and cellular debris seen at the site of viral replication. They are
useful in detection of viral infections with their distinct size, shape, location and staining
properties. They can be seen under light microscope.
5) Enumerate various inclusion bodies and indicate the viruses producing them.
Inclusion bodies Viruses
Negri bodies Rabies virus
Lipschultz body HSV
Torres body Yellow fever virus
Owl’s eye appearance body CMV
Paschen body Variola virus
56
6) What is the principle of Polymerase Chain Reaction (PCR)? Discuss its applications.
Principle of
PCR
It is a nucleic acid amplification technique used in diagnostic microbiology. Millions
of copies of nucleic acid are produced after PCR. It involves DNA extraction from
the organism, amplification of the extracted DNA and gel electrophoresis of the
amplified products. Thermocycler and polymerase enzyme are used.
Applications Detection of pathogens in clinical specimens
Detection of drug resistance in the pathogens
Detection of genetic diseases
Assessment

Total score /15
57
diagnosis of parasitic diseases
Competency
MI 1.2: Perform and identify the different causative agents of infectious diseases by Gram Stain,
ZN stain and stool routine microscopy
 Describe general steps in the laboratory diagnosis of parasitic infections
 Discuss the types of clinical specimen used in the laboratory diagnosis of parasitic
infections
 Diagnose the underlying parasitic infection based on the history and clinical symptoms
and suggest the suitable laboratory test in the given case.
 Perform saline and iodine mount from the given stool sample and identify various
morphological forms of parasites.
 Identify various parasitic forms in the peripheral smear.
 Describe the role of immunological tests in the diagnosis of parasitic diseases.
Exercise 9:
1) Describe general steps in the laboratory diagnosis of parasitic infections

Microscopic examination of the clinical specimens like feces, blood, CSF,etc.
Detection of parasite specific antibodies in serum
Detection of parasite specific antigen
Detection of parasite specific nucleic acid
Culture, imaging, xenodiagnosis, animal pathogenecity, intradermal skin tests.
2) Describe the collection of stool specimen for parasitic examination

It is collected before starting antiparasitic drugs, just before the onset of symptoms, at least 3
times on alternate days [ 6 times for intestinal amoebiasis ]. It is collected in a sterile,
leakproof, wide mouthed container with a screw cap.
58
3) Enumerate different types of clinical specimen (other than stool) used for diagnosis of
parasitic infections and indicate the clinical conditions.
Specimen Clinical condition/s
Peripheral blood Malaria, Filariasis
Liver aspirate Hepatic amoebiasis
CSF Encephalitis
Vaginal discharge Trichomoniasis
4) Draw the colored labeled diagrams of common parasitic form seen in stool
Fig. 45.2A Fig 45.6 A
Fig.45.10A Fig.45.10C
59
5) Draw the colored labeled diagrams of common parasitic form seen in peripheral blood smear
Fig.35.3 Early Gametocyte of P .falciparum Fig.37.4A
6) Enumerate immunological tests used in the diagnosis of parasitic infections and their
applications.
Immunological tests Applications
ELISA Detection of antibody in amoebic liver abscess
ICT Detection of antibody in visceral leishmaniasis
Sabin-Feldman dye test Detection of antibody in toxoplasmosis
Western blot Detection of antibody in cysticercosis
ELISA Detection of antigen in amoebiasis
ICT Detection of antigen in malaria
Triage parasite panel Detection of antigens in Giardiasis, amoebiasis,

cryptosporidiasis
60
Assessment

session
Total score /15
61
Exercise 9.2: Stool Examination
A lady aged 22 years complains of abdominal discomfort and altered bowel habits since
one week. She has submitted stool sample. Perform stool examination to look for parasitic
elements. Record your observations and interpret the results.
Fig.45.2 B
Observations
Round, 12 – 15 microns in size, contains 4 nuclei, thick cell wall
cyst is seen.
Inference
Quadrinucleated cyst of Entamoeba histolytica. Indicates
intestinal amoebiasis
62
63
Skill Certification
Competency No.: MI 1.2 Competency: Stool Examination (1)
(05) Scored
Focusses the preparation appropriately 01

writes the report
Score


64
diagnosis of fungal diseases
Competency
MI1.1: Describe the different causative agents of Infectious diseases and the methods used in
 Recognize the need for laboratory diagnosis of viral infections
 Enumerate major fungal pathogens and the infections/diseases caused by them
 Identify major fungal pathogens based on the morphology (yeast, molds etc.)
 Discuss general steps involved in the diagnosis of fungal infections
 Enumerate the common fungal culture media
 Enlist common tests/methods for fungal identification.
 Compare and contrast the laboratory methods used in the diagnosis of fungal infections
with bacterial infections
Exercise 10:
1) Explain the need for laboratory diagnosis of fungal infections

Fungal infections cause problems mainly in immunocompromised individuals. Sometimes
the problems can be chronic in nature. Therefore Detection of the causative fungal agent is
essential in planning treatment of the infection accurately.
2) Enumerate major fungal pathogens and the infections/diseases caused by them
Fungi Infection/Disease caused Fungi Infection/Disease caused
Mucor Zygomycosis Cryptococcus Cryptococcosis

neoformans
Dermatophytes Tinea Rhinosporidium Rhinosporidiosis

seeberi
Madurella Mycetoma Pneumocystis Pneumocystosis

mycetomatis jirovecii
Candida Candidiasis Aspergillus Aspergillosis

albicans flavus
65
3) Draw colored labeled diagrams of major morphological classes of fungi Fig.6.1 A to E
4) Write the common fungal culture media and their uses
Culture media Uses
Sabouraud’s dextrose agar Allows nonfastidious fungi to grow
CHROM agar Helps in species identification of Candida
Niger seed agar Selective medium for Cryptococcus
BHI agar Allows fastidious fungi to grow
66
5) Write the common fungal identification tests/methods and their uses
Identification tests/methods Uses
Microscopy Visualizes fungal elements in clinical

specimens
Culture Cultivation of fungi
Antigen and antibody detection Rapid detection
MALDI-TOF Accurate detection in short time
PCR Accurate detection short time
6) Draw a labeled diagram of the microscopic appearance of a dermatophyte in KOH mount.
Fig.6.2A
7) Draw a labeled diagram of the microscopic appearance of a mold in LPCB mount.
Fig.58.4B
67
8) Write the differences between laboratory methods used for the diagnosis of fungal infections
and bacterial infections
Fungal diagnostic methods Bacterial diagnostic methods
Antibiotics are used in culture media to Not used

prevent bacterial growth
Culture usually takes few days to few Usually grow in few days
weeks
Immunodiagnosis available for few Available for many infections

infections
Biochemical test available for few Available for many infections

infections
Assessment

session
Total score /15
68
11. Hospital infection control I: Hand
hygiene, PPE (Donning & Doffing)
Competencies
 MI8.5: Define Healthcare Associated Infections (HAI) and enumerate the types. Discuss the
factors that contribute to the development of HAI and the methods for prevention
 MI8.6: Describe the basics of Infection control
 MI8.7: Demonstrate Infection control practices and use of Personal Protective Equipment
 MI8.8: Describe the methods used and significance of assessing the microbial contamination
of food, water and air
Specific Learning Objectives; At the end the session, the students shall be able to,
• Describe the need for performing hand hygiene
• Enumerate types of hand hygiene based on the materials used in each type
• Choose appropriate method of hand hygiene in a given situation
• Perform WHO’s my five moments of hand hygiene
• Describe indications and procedure of using PPE
• Choose and perform the personal protective equipment (PPE) in a given simulated
healthcare setting
Exercise 11.1:
1) Perform donning and doffing of various personal protective equipment (PPE) used in in
healthcare setting such as gloves, 3-ply mask, N95 respirator, gown and goggles in the
recommended sequence
Steps of mask donning (wearing).

Fig.21.6
69
Steps of mask doffing (removal) Fig.21.7
Steps of gown donning (wearing). Fig.21.8

Steps of gown doffing (removal)
Fig.21.9
Exercise 11.2:
1. Discuss WHO’s my five moments for hand hygiene.
Before touching a patient
Before a procedure
70
After a procedure or body fluid exposure risk
After touching a patient
After touching patient’s surroundings
2. What are the products used for hand hygiene?
70 – 80% ethyl alcohol
0.5 – 4% chlorhexidine
Ordinary soap and water
3. What is relationship between hand hygiene and glove use?
Glove is a not a substitute for hand hygiene. Hand hygiene performed before
gloves use and hand wash done after gloves use. No hand hygiene done on the
gloved hand.
4. What is fit check and discuss its significance?
It is done after wearing N95 respirator. It ensure whether N95 respirator is
properly fitted. No clinical activity is done unless the respirator is properly fitted.
This checks sealing, positive pressure seal, negative pressure seal.
5. What are the precautions to be followed while removing a mask?
Do not touch front part of the mask
Untie the lower knot first, then the upper knot and remove the mask by holding its
straps, without touching the front
Hand wash after removal
6. Name indications where hand wash is necessary and hand rub may not be
effective
When the hands are visibly soiled with blood, excreta, pus, etc.
Before and after eating
After going to toilet
Before and after shift of the duty
When giving care to a patient with diarrhea
Assessment
71
session
Total score /15
72
Exercise 11.3: Choose appropriate PPE and demonstrate donning and doffing of the
selected PPE for Blood spill management. Perform hand hygiene wherever appropriate
Checklist for Assessment of Certification Skill
Hand hygiene

(Score=0)
1 Removes all hand accessories ( finger ringwrist 1.0
watch. etc.) (0.5) &Applies sufficient amount of
soap/hand wash /hand rub (0.5)
3 Rubs back of palm on both sides (0.5 + 0.5) 1.0
4 Follows rotational rubbing of thumb on both sides 1.0

(0.5 + 0.5)
5 Rubs back of fingers on palm on both sides (0.5 +0.5) 1.0
7 Rubs nails on palms on both sides (0.5 + 0.5) 1.0

Total score 10 /10

73

(Score=0)
Donning Glove:1 Wears by touching and pulling only the 1.0
edge of the cuff
Glove:2 a) Wears by pulling the external surface 1.0

of second glove by
Doffing Glove:1 Removes first glove by using the other 1.0

gloved hand, grasps the palm area of the
first glove & peels it off.
Glove:2 a) Holds the removed glove in the other 1.0

gloved hand (0.5) and
b) slides fingers of the ungloved hand
under the other glove at wrist and peels
off second glove over first glove. (0.5)

b) Performs hand hygiene after gloves removal
(0.5)
Total score 5.0 /05
74
Sequential Donning (wearing) of PPE
Student’s performance Score YES NO

(Score=0)
Performs proper hand hygiene 1.0
Wears the gown first in sequence 0.25
Wears the gown by fully covering torso from neck to 0.5

knees, arms to end of wrists, and wraps around the back
and fasten it at the waist and back of neck
Wears the mask second in sequence 0.25
Pulls the straps tight and pulls the mask to below chin and 0.5
then applies knots.
Presses on the nasal bridge part of the mask to seal tightly 0.5
and for N95 respirator, performs fit check.
Wears the goggles/face shield third in sequence 0.25
Wears the goggles/face shield by touching the front part 0.5

only
Wears the gloves last in sequence 0.25
Glove:1 Wears by touching and pulling only the edge of 0.5

the cuff

second glove bythe finger of gloved hand (0.25)
Total score 5.0 /05

Sequential Doffing (removal) of PPE
75
(Score=0)
Removes the gloves first 0.5
Does not touch outside of the gloves (contaminated) 0.5
Glove:1 Removes first glove by using the other gloved hand, 0.5
grasps the palm area of the first glove & peels it off.
Glove:2 a) Holds the removed glove in the other gloved hand 0.5
(0.25) and
b) slides fingers of the ungloved hand under the other
glove at wrist and peels off second glove over first glove.
(0.25)
Discards the gloves into red bin 0.5
Performs hand hygiene after gloves removal 0.5
Removes face shield/ goggles second in sequence 0.5
Removes face shield/ goggles by touching the sides only and 0.5
bending forward
Discards face shield/ goggles into red bin 0.5
Does not touch the front part of the gown while removing 0.5
Unfastens the gown ties, taking care that sleeves do not touch the 0.5
body while reaching for ties
Pulls the gown away from neck and shoulders, by touching inside 0.5
of gown only
Turn the gown inside out and rolls it into a bundle and discards 0.5
Discards the gown into yellowbin 0.5
Performs hand hygiene after removal 0.5
Takes off mask fourth in sequence 0.5
Does not touch front part of the mask. 0.5
Unties the lower knot first, then the upper knot and removes the
mask by holding its straps, without touching the front
Discards the mask into yellowbin 0.5
Performs hand hygiene at the last after removal of all PPE 0.5
Total score 10 /10
76
12.Hospital infection control II:
Sterilization & Disinfection, Biomedical
waste management, Needle stick injuries
Competencies
• MI1.4 Classify and describe the different methods of sterilization and disinfection.
Discuss the application of the different methods in the laboratory, in clinical and surgical
practice
• MI1.5 Choose the most appropriate method of sterilization and disinfection to be used in
specific situations in the laboratory, in clinical and surgical practice
• MI8.6: Describe the basics of Infection control
• MI8.7: Demonstrate Infection control practices
• MI8.8: Describe the methods used and significance of assessing the microbial
contamination of food, water and air
Specific Learning Objectives

• Classify the common sterilants and disinfectants used in healthcare settings and describe
their clinical applications
• Discuss the role of central sterile supply department (CSSD)in healthcare settings
• Describe the principles of segregation of biomedical waste
• Demonstrate appropriate segregation of biomedical waste (simulated)
• Describe the steps of blood spill management
• Describe the principles of respiratory hygiene and cough etiquette
• Describe the methods used and significance of assessing the microbial contamination of
food, water and air
• Interpret the results of common tests performed to assess microbiological contamination
of food, water and air
77
Exercise 12.1:
1) List the clinical applications of steam sterilizer in a healthcare setting

Used to sterilize surgical instruments, anesthetic equipment, dental instruments, implanted
medical devises, surgical drapes, linen, culture media, biomedical waste and sharps.
2) List the clinical applications of ethylene oxide sterilizer in a healthcare setting
Used to sterilize heart lung machine components, sutures, catheters, stents, respirators, dental
equipment, devices with electronic conmponents, assembled complex devices, multi-lumen
tubings.
3) List the clinical applications of plasma sterilizer in a healthcare setting
Used in CSSD to sterilize plastics, electrical devices, arthroscope, micro and vascular
instruments, spine sets and laparoscope.
4) Discuss the unidirectional workflow present in central sterile supply department (CSSD)
It consists of 4 unidirectional zones starting from a unsterile area to a sterile area separated
by a physical barrier.
Decontamination area
Packaging area
Sterilizing area
Sterile storage area
5) List the clinical applications of membrane filter in a healthcare setting
They filter air and water
6) List the clinical applications of dry heat sterilizer in a healthcare setting
Used to sterilize glass wares, powders, petroleum products, sharp instruments which do not
withstand moist heat
7. List the clinical applications of glutaraldehyde in a healthcare setting

It is a high level disinfectant used to sterilize endoscopes and cystoscopes
8. List the clinical applications of alcohol in a healthcare setting

60 – 80% of alcohol is used in hand rub, disinfection of thermometers, rubber stoppers, hubs of
central lines, stethoscopes, ventilators, manual ventilation bags, ultrasound machines,etc.
9. List the clinical applications of phenol in a healthcare setting

78
It is used as a disinfectant in the name of crisol or lysol in cleaning environmental
surfaces, bedside tables, bedrails, laboratory surfaces, noncritical medical devices and
sputum samples. A phenol compound such as Chloroxylenol is used as antiseptic.
10. List the clinical applications of povidone iodine in a healthcare setting
It is available as betadine and used as antiseptics at various concentrations for various purposes.
11. List the clinical applications of Quaternary ammonium compound (QAC)in a healthcare
setting
They are used in disinfection and cleaning of environmental surfaces such as floors, furniture,
walls, blood pressure cuffs.
12. Choose appropriate chemical disinfectant/sterilant in various clinical/laboratory situation

S.No. Clinical/Laboratory situation Disinfectant/Sterilant used
1. Surgical site preparation before surgery Povidone iodine
2. Laboratory surface before and after work Phenol
3. Floors in hospital at regular intervals QAC
4. Endoscopes after use Gluteraldehyde
5. Hand rub before surgery Alcohol
6. Glassware to prepare culture media Dry heat sterilizer
7. Air in operarion theaters Membrane filters
13. List the common biomedical waste items generated in healthcare setting that are segregated
in yellow bag.
Anatomical waste, soiled waste, expired/discarded medicines, chemical solid wasts, chemical
liquid waste, discarded linen waste, laboratory waste, blood bags, live vaccines.
14. List the common biomedical waste items generated in healthcare setting that are
segregated in red bag.
Infectious plastic waste such as tubing, bottles, intravenous tubes and sets, catheters, urine bags,
syringes without needles, vacutainers, gloves, aprons, goggles.
15. List the common biomedical waste items generated in healthcare setting that are
segregated in puncture-proof white/translucent container.
79
Needles, syringes with fixed needles, needles from needle tip cutter, scalpels, blades,
any other contaminated sharp object.
16. List the common biomedical waste items generated in healthcare setting that are segregated
in puncture-proof blue container.
Metallic body implants like dental implants, other body implants and plates. Glassware which is
broken or discarded, medicine vials and ampouls.
16. Describe briefly the steps of management of blood spill in correct sequence ?
Attend immediately. Mark the area. Place the wet floor signage. Wear gloves and
gown. Confine the spill and wipe immediately with an absorbent cloth and Dispose it
as infectious waste. Then clean with freshly prepared hypochlorite with a contact time
of at least 10 minutes. Finally, clean with water.
17. Name the components of the respiratory hygiene and cough etiquette?
Do not directly cough/sneeze on hands. Use your sleeve/inner elbow while coughing.
Wear surgical mask to limit the spread. Turn your head away from others and use tissue
while coughing/sneezing. Do hand hygiene after coughing/sneezing.
18. Name the methods used for assessing the microbial contamination of water?
Multiple tube method
Membrane filtration method
Presence/absence method
19. Name the methods used or assessing the microbial contamination of air?
Passive monitoring/settle plate method
Active monitoring/slit sampler method
Air particle counters
20. Enumerate indications for performing environmental surveillance in a healthcare setting?
Outbreak investigation
To evaluate the change in infection control practice
Construction
Research purpose
80
Exercise 12.2: Needle stick injury.
Analyze the case scenarios provided and discuss measures to be taken to prevent transmission of
infection(s) due to needle stick injury in each of them.
A. A 32-year-old staff working in biomedical waste department reported to HICC with

complaints of needle stick injury while segregating a yellow bag. The incident happened 18
hours back. He did not perform any first aid measures at the time of injury. He has not
received any hepatitis B vaccination before. Discuss the post-exposure prophylaxis measures
that should be undertaken.
HBIG – 1 dose given immediately
Start HBV vaccine course simultaneously at a different site
Give First dose of PEP for HIV
Do Tests for BBVs
Take Informed consent and give counseling
Document the exposure
Plan follow up testing for BBVs and plan management accordingly
B. A 28-year-old medicine resident reported to HICC with complaints of needle stick injury on
his left thumb while recapping the needle. The incident happened 1 hr back. On enquiry, he
mentioned that he had immediately washed his finger with running tap water for 2 minutes.
The source sample was tested and was found to be positive for hepatitis B surface antigen
and reactive for HIV antibodies. The resident had received a complete course (one series) of
hepatitis B vaccine 10 years back, following which he had an anti-HBs titer of 550 mIU/mL.
Discuss the post-exposure prophylaxis measures that should be undertaken.
Give First dose of PEP for HIV immediately

Do Tests for BBVs
Give PEP for HIV for 28 days with 3 drugs in combination [ tenofovir+lopinavir+ritonavir ]
81
C. A 41-year-old surgeon reported to HICC with complaints of surgical blade injury on his left
thumb, 3 hr back. The source sample was tested positive for hepatitis B surface antigen, but
negative for HIV antibodies and HCV antibodies. The surgeon had received two doses of
hepatitis B vaccine 2 years back. Discuss the post-exposure prophylaxis measures that should
be undertaken
HBIG – 1 dose given immediately

Complete HBV vaccine course simultaneously at a different site
Do Tests for BBVs
Assessment

session
Total score /15
82
Block II
Bloodstream and Cardiovascular System
Infections
83
13. Laboratory diagnosis of Rheumatic
heart disease, Infective Endocarditis and
Sepsis
Competencies
• MI2.1: Describe the etiologic agents in rheumatic fever and their diagnosis
• MI2.2:Describe the classification etio-pathogenesis, clinical features and discuss the
diagnostic modalities of Infective endocarditis
• MI2.3:Identify the microbial agents causing Rheumatic heart disease & infective
endocarditis

• Enumerate the etiologic agents in rheumatic fever
• Describe the methods of laboratory diagnosis rheumatic fever
• Enumerate the etiologic agents in infective endocarditis
• Describe methods of laboratory diagnosis of infective endocarditis
84
Exercise 13.1:
Clinical case: A case of fever, weakness and cardiac valvularlesions
1. What is the clinical diagnosis and how you arrived at it?

Infective endocarditis
2. Name the common etiological agents of this clinical condition
Staphylococcus aureus. Staphylococcus epidermidis. Viridians Streptococci. Enterococcus.
3.Describe the diagnostic criteria used for this condition.

Modified Duke criteria are used in diagnosis. 2 major or 3 minor+1 major or 5 minor
criteria are required for diagnosis. Positive blood culture and evidence of endocardial
involvement are the major criteria. Predisposition, fever, vascular phenomena,
immunologic phenomena, microbiologic evidence are the 5 minor criteria.
4. Describe the procedure to collect specimen for this clinical condition
Blood sample collected to isolate the organism. 2 blood culture sets [ a set is a pair of
blood culture bottles collected from different venepuncture sites ] should be collected
at an interval of >12 hours between first and second set.
5. Suggest treatment for this condition
Antibiotics are given depending upon the etiology.
85
Exercise 13.2:
Clinical case: A case of migrating joint pains, subcutaneous nodules and murmur

Migrating joint pains, subcutaneous nodules and murmur are the 3 major modified Jones
criteria seen in this patient. Presence of 2 major criteria are sufficient enough to diagnose
the condition as Rheumatic heart disease.
2. Name the etiological agent of this clinical condition and the underlying pathogenesis?
Streptococcus pyogenes. Pathogenesis is due to molecular mimicry resulting in autoimmune
disease. Antibodies targeted against Streptococcal antigens cross react with human tissue
antigens resulting in damage.
3.Describe the diagnostic criteria used for this clinical condition.
Diagnosis is made based on modified Jones major and minor criteria. Carditis, chorea, arthritis,
erythema marginaum, subcutaneous nodules are the 5 major criteria. Polyarthralgia,
hyperpyrexia, rise in ESR and or rise in CRP, prolonged PR interval are the 4 minor criteria. 2
major or 1minor+1major criteria are taken into consideration for diagnosis.
4. Describe briefly the prevention of recurrence of such episodes

Long term penicillin prophylaxis given every 4 weeks. Erythromycin 250mg twice a day can be
given as an alternative drug if patient is allergic to penicillin. It is given for 10 years depending
on the age.
86
Assessment

session
Total score /15
87
Exercise 13.3:
Clinical case: A case with fever, low BP, increased respiratory rate, alerted mentation and
positive blood culture

The patient is in Sepsis as suggested by the clinical presentation.
2. Name scoring system used to assess the severity of infection and extent of organ failure
SOFA score. Sepsis related organ failure assessment.
3. Name the common etiological agents of this clinical condition
Escherichia coli. Klebsiella pneumoniae. Pseudomonas aeruginosa. Staphylococcus aureus.
Haemophilus influenza type b. Salmonella typhi. CONS.
4. Describe the procedure to collect the specimen
Blood sample collected to detect the pathogen before initiating antibiotic therapy. It is collected
in strict aseptic condition. Blood collected in pairs from 2 different venepuncture sites. Skin
asepsis is done by 70% isopropyl alcohol followed by chlorhexidine. Air dry. a sterile syringe
and a needle is used to draw 10 ml blood in adult and 2 ml in child. At least 2 blood culture sets
taken, 1 for aerobes and the other for anaerobes. The blood sample is transported to lab
immediately and stored at 37 degrees C in incubator.
5. Describe the laboratory diagnosis of this clinical condition.
Blood sample collection.

Blood culture in conventional method.
The isolated pathogen is identified by Gram stain, colony morphology, biochemical
reactions.
Then antimicrobial susceptibility test is done to guide appropriate therapy.
Identification of the bacterial pathogen and AST can be done by automated methods
such as MALDI-TOF or VITEK
88
6. A heat-fixed smear made from the flagged blood culture bottle is provided. Perform Gram
staining, focus under microscope, record your observations and interpret the results
fig.28.2A
Observations Violet colored, round shaped bacteria seen in chains

Inference Streptococci
Example Viridians streptococci - eg. S.sanguis
Skill Assessment
Competency No.: MI 1.2 Competency: Gram Stain (2)
(05) Scored
Draws colored labeled diagram of the microscopic field and writes 01 (0.5+0.5)
the report
Score


89
14. Laboratory diagnosis of Brucellosis,

Leptospirosis, Dengue fever &
Chikungunya, scrub typhus and Systemic
candidiasis
Competencies
• MI8.1: Enumerate the microbial agents and their vectors causing Zoonotic diseases.
Describe the morphology, mode of transmission, pathogenesis and discuss the clinical
course, laboratory diagnosis and prevention
• IM4.3: Discuss and describe the common causes, pathophysiology and manifestations of
Dengue, Chikungunya, and Typhus fever
• Enumerate the microbial agents and their vectors causing Zoonotic diseases
• Describe the clinical manifestations and laboratory diagnosis of brucellosis
• Describe the clinical manifestations and laboratory diagnosis of leptospirosis
• Describe the clinical manifestations and laboratory diagnosis of scrub typhus
• Describe the clinical manifestations and laboratory diagnosis of dengue haemorrhagic
fever
• Describe the laboratory diagnosis of systemic candidiasis
• Interpret the laboratory tests results to diagnose common zoonotic diseases
Exercise 14.1:
Clinical case: A case of intermittent fever, organomegaly, positive blood culture
1) What is the clinical diagnosis and how you arrived at it?

Brucellosis. Undulating fever, hepatosplenomegaly and positive blood culture are typical of
brucellosis.
2) Explain the mode of transmission of this disease
It is zoonotic disease transmitted to man from infected animals like sheep, goat, camel, cattle,
buffalo and pigs.
90
3) Interpret the results of the laboratory test displayed
Small Gram negative coccobacilli. Catalase positive. Oxidase positive. Urease positive.
4) Suggest other microbiological tests for diagnosis of this condition
Standard agglutination test or ELISA which detects antibodies to Brucella antigens.
PCR assay.
MALDI-TOF or VITEK systems.
Rifampin + doxycycline for 6 weeks.
Exercise 14.2:
Clinical case: A rice-field worker with jaundice, organomegaly, oliguria and fever
1. Identify the clinical condition and justify your answer
Leptospirosis- Weil’s disease. History of Rice field worker, jaundice, oliguria, fever are
suggestive of the disease.
2. Explain the mode of transmission of this disease
Zoonotic disease transmitted from infected animals like rats, dogs, pigs, cattle.
3. What are the common clinical manifestations observed?
High fever, jaundice, hemorrhages, renal failure.
4.What are the various modalities of laboratory diagnosis?
Blood sample collected. Dark ground Microscopy with silver impregnation. Culture. ELISA to
detect antibody. PCR to detect specific genes.
5. How will you treat this condition?
Penicillin 1.5 million units given intravenously, 4 times a day for 7 days. In penicillin allergy
cases ceftriaxone or cefotaxime are used.
91
Assessment

session
Total score /15
92
Exercise 14.3:
Clinical case: A case of skin rashes with eschar
Scrub typhus. The classic presentation is triad of eschar, regional lymphadenopathy,

maculopapular skin rash.
2. What is the etiological agent and how it is transmitted?
It is caused by Orientia tsutsugamushi which is transmitted to man by the bite of mite called
Leptotrombidium deliensis.
3. What are the clinical manifestations seen in this condition?
Apart from classid triad, fever, headache, myalgia, cough and GIT symptoms may be seen.
4. What are the various modalities of laboratory diagnosis?

Heterophile antibody detection by Weil Felix reaction
Specific antibody detection by IFA or ELISA
PCR

Doxycycline is the drug of choice.
93
Exercise 14.4:
Clinical case: A case of fever, joint pain, rash and thrombocytopenia
Dengue haemorrhagic fever. Fever, joint pains, maculopapular rash, headache, organomegaly,
thrombocytopenia, haemorrhages are the clinical manifestations of DHF.
2. List the viruses that cause hemorrhagic fever.
Dengue virus, yellow fever virus, KFD virus, Ebola virus and Marburg virus

Dengue fever, dengue hemorrhagic fever, dengue shock syndrome

NS1 antigen detection by ICT/ELISA
IgM and IgG antibody detection by ELISA/ICT
Virus isolation in mosquito cell line
RTPCR
Symptomatic and supportive treatment given. Replacement of plasma/platelets/electrolytes that
are lost. Correction of metabolic disturbance.
94
Exercise 14.5:
Clinical case: An HIV patient with fever and hypotension, budding yeast cells in blood
culture
1. What is the clinical diagnosis and the likely etiological agent?
Systemic candidiasis. Candida albicans.
2. Name the risk factors predisposing this clinical condition.

Immune suppression – eg,AIDS. Uncontrolled diabetes mellitus. Chronic renal failure. Terminal
cancer satge. Patient on immunosuppressive drugs. Extremes of age. Patient On steroids.
3. What are the other clinical manifestations caused by this organism?

UTI. Pulmonary candidiasis. Septicemia. Arthritis. Osteomyelitis. Meningitis.
Keratoconjunctivitis and endophthalmitis. Hepatosplenic candidiasis. Disseminated candidiasis.
Direct microscopy. Culture on SDA. Germ tube test. CHROMagar morphology. Carbohydrate
assimilation tests. MALDI-TOF. PCR. beta-d-glucan assay. Immunodiagnosis.
Antifungal agents like Liposomal amphotericin B or caspofungin or voriconazole.
95
Assessment

session
Total score /15
96
15.Laboratory diagnosis of Enteric fever
Competencies
• MI3.3: Describe the enteric fever pathogens and discuss the evolution of the clinical
course, the laboratory diagnosis of the diseases caused by them
• MI3.4: Identify the different modalities for diagnosis of enteric fever. Choose the
appropriate test related to the duration of illness
• Enumerate organisms causing enteric fever
• Describe the clinical manifestations of enteric fever
• Describe the laboratory diagnosis of enteric fever
• Choose appropriate microbiological investigation for diagnosis of enteric fever in
different periods of enteric fever
• Interpret the laboratory tests done for the detection of enteric fever pathogens
• Describe the treatment modalities of enteric fever
Exercise 15.1:
Clinical case: A case of step ladder fever and splenomegaly with positive blood culture

Enteric fever. Remittent fever, splenomegaly and positive blood culture are suggestive of enteric
fever.
2. How this disease is transmitted?
Ingestion of contaminated food and water.
Step ladder fever, rose spots, hepatosplenomegaly, epistaxis, relative bradycardia, headache,
nausea, vomiting. Complications like GIT bleeding and intestinal perforation can occur.
97
4. What are the various modalities of laboratory diagnosis in different periods of the disease?
___________________________________________________________
S.No. Period of disease Useful microbiological test
1 1st week Blood culture
2 2nd week Antibody detection by Widal test
3 3rd week Antibody detection by Widal test
4 4th week Stool culture
5 Carrier Stool and urine culture

Ceftriaxone is the drug of choice. Azithromycin is the alternative. Ciprofloxacin can be used
only, if found susceptible.
6. Interpret the Widal test results (Titers)
S.NO. S.Typhi S.Typhi S.Paratyphi S.Paratyphi Interpretation
O H AH BH
1 20 40 <20 <20 Past typhoid fever
2 160 320 <20 <20 Typhoid fever
3 320 40 640 <20 Paratyphoid A fever
4 160 40 <20 <20 Recent enteric fever
5 80 320 160 160 Post TAB vaccination
98
Assessment

session
Total score /15
99
16.Laboratory diagnosis of Malaria
Competencies
• MI2.5: Describe the etio-pathogenesis and discuss the clinical evolution and the
laboratory diagnosis of malaria
• MI2.6: Identify the causative agent of malaria

• Enumerate organisms causing malaria
• Describe pathogenesis of malaria
• Describe the clinical manifestations, laboratory diagnosis and treatment of different
types of malaria
• Describe and identify diagnostic forms of malarial parasites in peripheral blood smear
• Interpret laboratory tests to detect malarial parasites
Exercise 16.1:
Clinical case: A case of fever with chills and rigors, splenomegaly

Falciparum Malaria. Febrile paroxysm and splenomegaly are suggestive of malaria.
2. What is the host, infective form, and mode of transmission of the parasite?
Female anopheles mosquito is the definitive host. Sporozoites are the infective form. Man
aquires infection by the bite of mosquito.
3. What are the various complications seen?

Cerebral malaria, black water fever, algid malaria, hypoglycemia, septicemic malaria, jaundice,
anemia.
100
4. What are the various diagnostic modalities?
Peripheral blood smear examination. Fluorescence microscopy. Quantitative byffy coat
examination. Antigen detection. PCR. culture.
5 How will you treat this clinical condition?

Antimalarial drugs like chloroquine, primaquine, quinine are used dependind on severity,
species, resistance to drugs.
6.Draw labelled diagrams of common diagnostic forms of this parasite focused
Fig.35.3 draw early trophozoite and gametocyte forms of P.falciparum
101
Exercise 16.2:
Clinical case: A case of fever with chills and rigors with splenomegaly and anemia

Febrile paroxysm and splenomegaly are suggestive of vivax malaria. Anemia is a complication
of malaria with high parasitemia and low haemoglobin levels. It is a result of parasite destruction
of RBC, splenic removal of RBC, bone marrow suppression.
2. What is the host, infective form, and mode of transmissionof the parasite?
Female anopheles mosquito is the definitive host. Sporozoites are the infective form. Man
aquires infection by the bite of mosquito.
3. What is relapse and how it is different than recrudescence?

Recrudescence is commonly seen in P.falciparum and is due to persistence of drug resistant
parasites even after the completion of the treatment. Relapse is seen commonly in P.vivax and is
due to reactivation of dormant hypnozoites leading to initiation of erythrocytic cycle and relapse
of malaria.
4. What are the various diagnostic modalities?
Peripheral blood smear examination. Fluorescence microscopy. Quantitative byffy coat

examination. Antigen detection. PCR. culture.
5 How will you treat this clinical condition?

Antimalarial drugs like chloroquine, primaquine, quinine are used depending on severity,
species, resistance to drugs.
102
Assessment

session
Total score /15
103
17. Laboratory diagnosis of Filariasis and
Leishmaniasis
Competencies
• MI2.5: Describe the etio-pathogenesis and discuss the clinical evolution and the
laboratory diagnosis of kala-azar, filariasis and other common parasites prevalent in India
• MI2.6: Identify the causative agent of filariasis

• Describe the clinical manifestations, laboratory diagnosis and treatment of kala-azar
• Describe the clinical manifestations, laboratory diagnosis and treatment of lymphatic
filariasis
• Suggest appropriate sample collection method for laboratory diagnosis of filariasis and
kala-azar
• Identify the diagnostic forms of organisms causing kala-azar and filariasis
Exercise 17.1:
Clinical case: A case with fever, splenomegaly and hyperpigmentation

Visceral leishmaniasis. Fever, splenomegaly and hyperpigmentation are suggestive of
VL.
2. What is the host, infective form, diagnostic form, and mode of transmission of the parasite?
Man is vertebrate host. Sandfly is invertebrate host. Promastigotes are infective forms. Infection
transmitted by the bite of sandfly. Leishman - Donovan [LD] bodies inside macrophages are
diagnostic forms.
3. What are the other diagnostic tests to diagnose this condition?

Culture on NNN medium. Antibody detection by ELISA. Antigen detection in urine by LAT.
PCR.
104
Liposomal amphotericin B, miltefosin, paromomycin, pentavalent antimonials.
Exercise 17.2:
Clinical case: A case of fever and unilateral limb swelling

Acute lymphatic filariasis. Fever, edema are characteristic of the condition.
2. What are the etiological agents that can cause this clinical condition?
Wuchereria bancrofti
3. Draw a neat, labeled diagram of the etiological agent focused.
Fig.37.4A
105
4. How does man acquire this infection and what is the vector involved?
Infection transmitted by the bite of Culex quinquefasciatus.
5. Which is the infective stage of the parasite for man?
3rd stage filariform larva is the infective form.

6. What are the different modalities of diagnosis of this clinical condition?
Detection of microfilaria in peripheral blood by Giemsa stain. Antigen detectiob by ELISA.
Antibody detection by flow through assay. Filarial dance sign in lymphatics on ultrasonogram.
RT-PCR.
7. Describe the blood collection procedure to demonstrate the organism by microscopy

Peripheral Blood collected at night between 9pm and 4am. Blood can also be collected at day
time after DEC provocation test.
Diethylcarbamazine is the drug of choice. Albendazole is an alternative.
106
Assessment

session
Total score /15
107
18. Laboratory diagnosis of HIV infection
Competencies
• MI2.7: Describe the epidemiology, the etio-pathogenesis, evolution complications,
opportunistic infections, diagnosis, prevention and the principles of management of HIV
• MI8.12: Discuss confidentiality pertaining to patient identity in laboratory results

• Enumerate the modes of transmission of HIV infection
• Describe pathogenesis of HIV infection
• Describe the clinical manifestations, laboratory diagnosis and treatment of HIV
• Interpret the laboratory test results to diagnose HIV infection as per the NACO guidelines
• Enumerate and classify opportunistic infections in HIV/AIDS
• Discuss the importance of maintaining confidentiality pertaining to patient identity in
laboratory results
• Demonstrate maintenance of confidentiality pertaining to laboratory test results in a
simulated setting
Exercise 18.1:
Clinical case: A promiscuous man with chronic diarrhea and lymphadenopathy

HIV infection-AIDS. Promiscuous behavior [ multiple sex partners ], lymphadenopathy and
chronic diarrhea are suggestive of the diagnosis.
2. What are the various modes of transmission of the etiological agent?
Blood transfusion. Parent to child. Sexual intercourse. Injection drug abuse. Needle stick
exposure.
108
3. What are the different modalities of diagnosis of this clinical condition?
P24 antigen detection by ELISA or rapid ICT tests. Antibodies detection by ELISA and rapid
ICT tests. Reverse transcriptase PCR. Western blot assay. Viral culture.
4. Which are various opportunistic infections that can occur in this patient?
Candida oral thrush, Cryptococcal meningitis, Pneumocystis pneumonia, Tuberculosis,
Cryptosporidium diarrhea, Toxoplasma encephalitis, CMV retinitis, Strongyloides diarrhea
Antiretroviral drugs used as per NACO guidelines in combinations called as HAART. Tenofovir,
efavirenz, lamivudine are used in combination as first line treatment.
6. Discuss confidentiality pertaining to patient identity in laboratory results.
It is a part of the professional secrecy, where a patient’s personal health information is not shared
with others without the patient’s consent. The person with the HIV has the right to privacy, and
the right to exercise informed consent in all decisions about his disclosure of HIV status except
in circumstances when disclosure to another person is required by the law or ethical or health
considerations.
7. What is NACO strategy I and where it is applied? See Page.333.
It is done to screen blood donors. It is done with 1 test. If found reactive, then the unit of blood is
destroyed.
8. What is NACO strategy II and where it is applied?
Strategy IIA: It is done to screen population to estimate prevalence of HIV infection at a given
area. It is done with 2 tests. Negative result of first test is taken as HIV negative. Positive Result
of the first test should be confirmed with second test. If the second test result is negative, then it
is reported as HIV negative. Positive result in both the tests is reported as HIV positive.
Strategy IIB: It is done diagnose HIV in symptomatic individuals. It is done with 2 or 3 tests.
Negative result of first test is taken as negative. Positive Result of the first test should be
confirmed with second test. Positive results in both the tests is reported as HIV positive with post
test counseling. If the second test result is negative, then a 3 rd test is done for confirmation. If 3 rd
test result is also negative then report as HIV negative. If 3 rd test result is positive then report as
HIV indeterminate.
9. What is NACO strategy III and where it is applied?
It is done to detect HIV infection in asymptomatic HIV patients, antenatal screening and
screening of patients waiting for surgeries. It is done with 3 tests. Positive result in all 3 tests is
reported as HIV positive with post test counseling. Negative result in first test is taken as
negative. Positive result in first test should be confirmed by 2nd and 3rd tests. Negative result in
second test should be followed by 3rd test. Negative results in both 2nd and 3rd tests is taken as
negative. Positive result in 3rd test is taken as indeterminate. Positive result in 2nd test is followed
by 3rd test. Negative in 3rd test is taken as indeterminate.
AETCOM Exercise
Exercise 18.2:
109
Confidentiality pertaining to patient's identity in lab result
Competency
MI 8.14 Demonstrate confidentiality pertaining to patient identity in laboratory results
Specific learning objectives

At the end of the module students should be able to
 Describe confidentiality of patient identity in relation with laboratory investigations
 Identify the routine events in testing and reporting of clinical samples where the
confidentiality of patient identity is applicable.
 Identify the ways to rectify breach in confidentiality in patient identity in relation with
laboratory investigations
 Discuss and apply with justification situations/reasons where breach in confidentiality is
acceptable.
 Describe and demonstrate application of ethical principles and medico legal issues with
respect to confidentiality of patient identity in laboratory results
In a given situation/case scenario, identify the events that show disrespect / breach in
confidentiality to the clinical sample/ data and suggest ways to rectify them.
1) A literate HIV patient getting embarrassed when she finds the test result mentioned on her
case file
It is a part of the professional secrecy, where a HIV status of the patient is not disclosed without
the patient’s consent. Patient with HIV has the right to privacy, and the right to exercise
informed consent in all decisions about the disclosure of his/her HIV status except in
circumstances when the disclosure to another person is required by law/ethical/health
considerations. In this situation, patient has to be educated that it is essential that health care
workers involved in patient care must be aware of her HIV status so that they can be aware of the
risk of infection associated in this process and take care accordingly. However, at the same time
confidentiality of the patient’s HIV status should be strictly restricted to health care workers
involved in patient care. An uncommon medical term like ‘Retro positive label’ is placed on case
file instead of ‘HIV positive’ so that HCW involved in patient care can understand that patient is
HIV positive and at the same time non medical people cannot understand the meaning of the
label.
110
2) Pre-surgical HIV test request received without pre test counseling from surgery ward
Normally pre test counseling before performing HIV test is done at ICTC. In this situation,
where surgery should be done on emergency basis the HIV test is carried out without pre test
counseling. The test is done as the patient is not in a situation to make personal decisions related
to HIV and also that, it is essential that health care workers involved in patient care must be
aware of patient’s HIV status so that they can be aware of the risk of infection associated in this
process and take care accordingly. However, at the same time confidentiality of the patient’s
HIV status should be strictly restricted to health care workers involved in patient care.
Counseling is considered later.
3)You are treating a Covid-19 positive patient and he does not want you to reveal his diagnosis
to others
In this situation, patient is identified with an alphanumeric code instead of his name. Details
are released in the media by the government authorities with the code and not disclosing the
patient identity. Labels are put on the house to identify and alert other people.
4) A nurse has needle stick injury while performing phlebotomy. The patient’s HIV and HBV
status is unknown. The patient is refusing to get tested for HIV
111
Patient should be advised counseling in order to make him understand the situation and the
importance of the test result in deciding the post exposure prophylaxis to be received by the
nurse.
Assessment

Total score /15
112
BLOCK III
Gastrointestinal and Hepatobiliary
Infections
113
19.Laboratory diagnosis of diarrhea
Competencies
 MI 1.2: Perform and identify the different causative agents of Infectious diseases by Gram
Stain, ZN stain and stool routine microscopy
 MI 3.1: Enumerate the microbial agents causing diarrhea and dysentery. Describe the
epidemiology, morphology, pathogenesis, clinical features and diagnostic modalities of these
agents
 MI 3.2: Identify the common etiologic agents of diarrhea and dysentery
Specific Learning Objectives,

At the end of the teaching-learning session the student shall be able to
 Prepare stool wet mount and describe the procedure to screen for parasitic elements
 Perform stool wet mount examination correctly and identify common ova, cyst and larvae
 Define and differentiate diarrhea and dysentery.
 Enumerate common microbial agents causing diarrhea. Classify them based on organism
type, common age group affected and duration of diarrhea
 Enumerate appropriate sample(s) for laboratory diagnosis of infectious diarrhea and describe
the correct method of collection and transportation of such samples to microbiology
laboratory
 Choose appropriate test(s) for microbiological diagnosis of diarrhea
 Analyze a case of diarrhea and suggest a plan of investigation(s) to confirm/rule out common
potential pathogens in that case
 Suggest appropriate sample and microbiological tests along with expected test results to
diagnose a case of suspected cholera
diagnose a case of suspected viral diarrhea
diagnose a case of suspected giardiasis
diagnose a case of suspected opportunistic intestinal parasitic infection
diagnose a case of diarrhea associated with antibiotic administration
 Describe procedure and applications of hanging drop preparation
 Draw and label common parasitic elements seen in stool sample of person suffering from
diarrhea
114
Exercise 19.1:
Clinical case: An outbreak of acute diarrhea with dehydration
Diagnosis is cholera
1. Identify the probable clinical diagnosis and causative agent. Justify your answer
Cholera. Vibrio cholerae. An outbreak with acute diarrhea with dehydration is suggestive of
cholera.
2. Name one simple and quick preliminary diagnostic test for this condition
Direct microscopy : comma shaped gram negative rods seen.

Hanging drop : darting motility seen.
Antigen detection by dipstick assay.
3. Suggest tests to confirm the most probable pathogen

Conventional Culture, biochemical tests, biotyping, serogrouping, serotyping.
Automated methods like MALDI-TOF and VITEK.
PCR.
4. The patient is in a village and nearest microbiology lab is 20 kms.from there. Describe
appropriate sample collection and transportation procedures for identifying the probable
pathogen. Suggest alternative options for the same
Freshly collected watery stool sample is transported in Venkatraman-Ramakrishnan medium

to the lab. Other transport media are alkaline salt transport medium or cary-Blair medium.
5. Based on the clinical features provided and laboratory tests displayed suggest appropriate
treatment in order of preference
Correct dehydration with fluids and electrolytes. Erythromycin given in severe cases.
6. Name 3 other possible pathogens responsible for such condition.

Escherichia coli. Non typhoidal Salmonellae. Bacillus cereus. Staphylococcus aureus.
115
Exercise 19.2:
Clinical case: A child with diarrhea
Diagnosis is Rota virus diarrhea
1. Identify the most probable pathogen in this case
Rota virus
2. Name other tests to confirm the laboratory diagnosis
Demonstration of virus in feces by immunoelectron microscopy. Antigen detection in

feces by ELISA. Viral RNA detection in feces by RTPCR.
3. Name the other organisms causing similar clinical manifestations
Sapoviruses. Adenoviruses types 40 and 41. Astroviruses.
4. List the laboratory tests to differentiate other pathogens causing this condition
Demonstration of virus in feces by immunoelectron microscopy. Antigen detection in

feces by ELISA. Viral RNA detection in feces by RTPCR.
5. Describe briefly specific preventive measures for this condition
Rotovac vaccine available. Improve hand hygiene and sanitation in the community.
6. A local doctor has prescribed Syp. Amoxycillin-clavulanic acid in appropriate dose and
frequency for 3 days. Is it justified?
No. the etiological agent is a virus. Antibiotics are not useful here. Fluids and electrolytes
are necessary to treat the condition.
116
Exercise 19.3:
Clinical case: Diarrhea in a person on antibiotic therapy
Diagnosis is pseudomembranous colitis/antibiotic associated diarrhea
1. Identify the probable etiological agent in this case

Clostridium difficile.
2. Describe the pathogenesis of this condition in brief
Antibiotics usage disrupts normal colonic flora, which enhances the susceptibility to
C.difficile infection.
3. Enumerate other risk factors associated with this clinical condition
Prolonged hospital stay, prolonged antimicrobial use, advanced age,
immunosuppression, cancer chemotherapy, malignancy, GIT surgeries.
4. Suggest principles of treating this condition
Vancomycin is the drug of choice.
117
Assessment

session
Total score /15
118
Exercise 19.4:
Clinical case: A case of chronic diarrhea, foul smelling stools with mucus
Diagnosis is chronic Giardiasis

1. Draw and label the two diagnostic forms of this organism
Fig.45.6A Fig.45.6 C
2. Suggest other samples and tests that can be done to diagnose this clinical condition
Duodenal aspirate collected by string test. Antigen detection by ELISA in stool sample. Nucleic
acid detection in stool sample.
3.Name the infective form of this organism and mode of transmission
Mature Cyst is the infective form. Mode of transmission is by ingestion of food or water
contaminated with cysts.
4.Enumerate complications of this infection
Urticaria, arthritis, anterior uveitis.
Tinidazole is the drug of choice.
119
Exercise 19.5:
Clinical case: An AIDS patient with persistent diarrhea
Diagnosis is Cryptosporidium diarrhea
1. Identify the etiological agent in this case

Cryptosporidium parvum
2. Enumerate other similar organisms causing this type of clinical presentation
Cyclospora cayetanensis. Cystoisospora belli.
3. List other laboratory tests to diagnose this condition
Stool sample is collected. Direct microscopy by acid fast stain and fluorescence stain.
Antigen detection by ELISA. Antibody detection by ELISA. PCR.
4. Describe the mode of transmission of this infection and suggest preventive measures
Ingestion of food or water contaminated with thick walled oocysts of the parasites. Improvement
in hand hygiene and sanitation measures help in reduction of transmission.
120
Assessment

session
Total score /15
121
Exercise 19.6: A 46-year-old lady presented with pain abdomen, intermittent diarrhea, loss of
weight, poor appetite and on-and-off fever since 3 weeks. CT scan of the abdomen showed
enlarged mesenteric lymph nodes and minimal ascites. CT guided lymph node aspiration was
done. Heat-fixed smear of the aspirate is provided. Perform suitable staining to demonstrate the
most probable pathogen, focus the smear under microscope, record your observations and
interpret the results
Diagnosis is mesenteric adenitis caused by Yersinia enterocolitica
Fig.41.1B
Gram stain
Observations Pink colored, rod shaped bacteria seen
Inference Gram negative bacilli
Example Yersinia enterocolitica
122
Skill Certification
(05) Scored

writes the report
Score


123

Clinical Case: A case of diarrhea and abdominal discomfort
Diagnosis is hymenolepiasis
1. Give appropriate container and advise the patient on sample collection
A sterile, screw capped, wide mouthed container is given to patient to collect his stool
sample
2. Perform stool examination with the sample provided and record your observations and
Fig.46.5A
Observations Oval shaped, 40 microns size, Non bile stained egg with polar
filaments seen
Inference Egg of Hymenolepis nana
124
3. Enumerate more sensitive laboratory tests to diagnose this condition
Stool concentration can be done to increase sensitivity
4. Describe source and mode of transmission of this infection
Eggs are the infective forms. Mode of transmission is by ingestion of food or water
contaminated with eggs.
Skill Assessment
(05) Scored

writes the report
Score


125
20. Laboratory diagnosis of dysentery
Competencies
 MI 1.2: Perform and identify the different causative agents of Infectiousdiseases by Gram
Stain, ZN stain and stool routine microscopy
 MI 3.1: Enumerate the microbial agents causing diarrhea and dysentery.Describe the
epidemiology, morphology, pathogenesis, clinicalfeatures and diagnostic modalities of these
agents

 Define and differentiate diarrhea and dysentery
 Enumerate common microbial agents causing dysentery. Classify them based on organism
type
 Enumerate appropriate sample(s) for laboratory diagnosis of dysentery and describe the
correct method of collection and transportation of such samples to microbiology laboratory
 Choose appropriate tests for microbiological diagnosis of dysentery
 Analyze a case of dysentery and suggest a plan of investigation(s) to confirm/rule out
common potential pathogens in that case
diagnose a case of suspected amoebic dysentery
diagnose a case of suspected bacillary dysentery
 Differentiate amoebic and bacillary dysentery based on macroscopic and microscopic
examination of stool sample
 Draw and label common parasitic elements seen in stool sample of person suffering from
dysentery
126
Exercise 20.1:
Clinical case: A case of frequent blood-stained stools
Diagnosis is bacillary dysentery caused by Shigella species
1. Record your observations of the tests displayed and interpret the results. Identify the
causative agent of this condition
Table 45.1
Small quantity of feces, more than 10 times per day, bright red, odourless, adherent to the
container. Gram negative bacilli and pus cells seen on Gram stain of stool sample.
2. Enumerate other organisms producing such clinical presentation

Shigella species. Campylobacter jejuni. Yersinia enterocolitica.
3. List laboratory investigations to confirm/refute other organisms listed above

Bacterial culture. Motility testing in hanging drop. Biochemical tests. Serotyping.
4. Enumerate complications of this condition
Toxic megacolo, intestinal perforation, rectal prolapsed, toxic encephalopathy, bacteremia.
Ciprofloxacin is the drug of choice. Fluids and electrolytes as required.
127
Assessment

session
Total score /15
128

Clinical case: A case of frequent dark red offensive-smelling stools
DIAGNOSIS is amoebic dysentery
1. Choose appropriate container and advise the patient regarding proper sample collection
Sterile, screw capped, wide mouthed container is given to patient to collect stool sample.
Stool sample collected 3 times on alternate days within 10 days period.
2.Perform stool examination with the sample provided and record your observation and
inference
Fig.45.2A
Observations Trophozoites seen
Inference Entamoeba histolytica
129
3. Enumerate other investigations to identify/confirm this pathogen
Stool culture. Stool antigen detection. PCR. Histology.
4. Enumerate other common pathogens responsible for dysentery
Bacteria like Shigella species. Campylobacter jejuni. Yersinia enterocolitica.
5. Differentiate dysentery caused by two common pathogens
Amoebic dysentery Bacillary/bacterial dysentery
More amount of stool excreted, Dark red. Small amount, bright red.
Offensive, not adherent to the container. Odorless, adherent to container.
Few pus cells seen. Numerous pus cells seen.
Charcot-Leyden crystal present. Charcot –Leyden crystal absent.
Cyst or trophozoite seen. Gram negative bacilli seen.
Bacterial culture negative. Bacterial culture positive.
130
Skill Assessment
(05) Scored

writes the report
Score


131
21. Laboratory diagnosis of intestinal
Helminthic infections
Competencies
 MI 1.2: Perform and identify the different causative agents of Infectious diseases by Gram
Stain, ZN stain and stool routine microscopy.
 MI 2.4: List the common microbial agents causing anemia. Describe the morphology, mode
of infection and discuss the pathogenesis, clinical course, diagnosis and prevention and
treatment of the and common microbial agents causing Anemia
 MI2.5: Describe the etio-pathogenesis and discuss the clinical evolution and the laboratory
diagnosis of kala-azar, malaria, filariasis and other common parasites prevalent in India
 MI 3.1: Enumerate the microbial agents causing diarrhea and dysentery
 Describe - the epidemiology, morphology, pathogenesis, clinicalfeatures and diagnostic
modalities of these agents
Specific Learning objectives
 List common microbial agents causing anemia
 Describe morphology of common helminths causing anemia
 Describe laboratory diagnosis of intestinal helminthic infections
 Identify the diagnostic forms (macroscopic and microscopic) of helminths causing anemia
 Enumerate common intestinal helminthic parasites prevalent in India
 Describe morphological forms including diagnostic forms of common intestinal parasites
 Suggest treatment and prevention of intestinal parasitic infections
 Define and differentiate diarrhea and dysentery.
 Enumerate common microbial agents causing diarrhea. Classify them based on organism
type, common age group affected and duration of diarrhea
 Enumerate appropriate sample(s) for laboratory diagnosis of infectious diarrhea and describe
the correct method of collection and transportation of such samples to microbiology
laboratory
 Choose appropriate test(s) for microbiological diagnosis of diarrhea
 Analyze a case of diarrhea and suggest a plan of investigation(s) to confirm/rule out common
potential pathogens in that case
 Perform stool examination to demonstrate diagnostic forms of intestinal parasites
 Draw neat labeled diagrams of diagnostic forms of intestinal parasites
132
Exercise 21.1:
Clinical case: A child who passes a motile fleshy material in stools
Diagnosis is ascariasis
1. Identify the material passed in stool by the child. Justify your answer
Adult ascaris worms. They are motile, cylindrical worms and fleshy in nature.
2. Enumerate other laboratory investigations to diagnose/confirm the infection
Egg detection in stool sample. PCR.
3. Describe briefly – mode of transmission, clinical manifestations and complications
Ingestion of eggs from contaminated soil, food, water is the mode of transmission.
Pneumonia, malnutrition, growth retardation, urticaria are clinical manifestations.
Intussusceptions, cholecystitis, pancreatitis are complications.
4. Draw neat labeled diagram of other diagnostic forms expected in stool microscopy in this
condition
Fig.46.18 A
133
Exercise 21.2:
Clinical Case: A case of abdominal pain and malnourishment
Diagnosis is trichuriasis

Trichuris trichiura
Egg detection in stool sample
3. Draw a neat labeled diagram of the diagnostic form
Fig.46.15A
4. Describe briefly the mode of transmission, clinical manifestations and complications

Mode of transmission :Ingestion of food or water contaminated with the embryonated egg.
Clinical manifestations :Abdomen pain, anorexia.
Complications :Dysentery, anemia, recurrent rectal prolapsed, malnutrition, growth retardation.
134
Assessment

session
Total score /15
135
Exercise 21.3:
Clinical case: An incidental stool finding in apparently normal individual
1) Perform stool examination to look for parasitic elements. Record your observations and
Fig.46.18 A
Fertilized egg
Observations Oval, bile stained, mamillated eggs seen
Inference Ascaris lumbricoides fertilized eggs
2) Enumerate other laboratory investigations to diagnose/confirm the infection

Antibody detection by ELISA in serum. PCR.
3) Describe briefly – mode of transmission and complications of this infection
Ingestion of food or water or soil contaminated with eggs.
Pneumonia, intussusception, cholecystitis, pancreatitis.
4) Suggest appropriate treatment

Albendazole 400mg once.
136
Skill Certification
(05) Scored
Focuses the preparation appropriately 01
the report
Score


137
Exercise 21.4:
Clinical case: A diabetic with persistent loose stools
Diagnosis is strongyloidiasis

Strongyloides ercoralis
Stool microscopy. Antibody detection by ELISA. Antigen detection
3. Draw neat labeled diagram of the diagnostic form
Fig.46.24 B
4. Describe mode of transmission

Skin penetration by larva [ during walking on barefoot ]
5. Enumerate complications of this infections and conditions predisposing to them

Hyperinfection syndrome and disseminated strongyloidiasis are the complications.
Predisposing factors are: glucocorticoid therapy, immunosuppressive conditions, cancer,
HTLV-1 infection, HIV infection.
138
Exercise 21.5:
Clinical case:A child with perianal itching
Enterobius vermicularis
2. Draw neat labeled diagram of the diagnostic form
Fig.46.16 C
3. Describe the ideal sample collection method and test done to diagnose this condition
Microscopy of perianal skin samples collected by cellophane tape method or by using NIH swab
method. Detection of eggs are diagnostic of infection.
4. State the utility of stool examination to diagnose this condition and Justify your answer
Eggs are rarely detected in stool sample as the female worm lays eggs on perianal skin and not in
rectum. Therefore detection of eggs in stool sample is not preferred method.
5. Suggest methods to prevent this infection
Improve personal hygiene, frequent hand wash, regular washing of bed clothes, keeping nails
short and clean.
139
Assessment

session
Total score /15
140
Exercise 21.6:
Clinical case:A case of anemia
1) Perform stool examination to look for parasitic elements. Record your observations and
Fig. 46.22 A
Observations Non bile stained, oval shaped egg with 4 blastomeres enclosed in
thin shell seen
Inference Egg of hook worm. Eg.Ancylostoma duodenale
2) Enumerate more sensitive laboratory investigations to diagnose/confirm the infection

Stool culture. PCR.
3) Describe briefly – mode of transmission, clinical manifestations and complications
Skin penetration by larvae during barefoot walking on dampen soil. Cutaneous lesions and
pneumonitis due to larval migration. Abdomen pain, diarrhea, eosinophilia, iron
deficiency anemia due to adult worms in intestine.
141
4) What can happen if the stool examination is performed after storing the sample at
room temperature for 1-2 days?
Eggs may hatch into rhabditiform larva and the larva has to be differentiated from the
larva of Strongyloides stercoralis. Larva of hook worm has longer mouth and small
genital primordium. Larva of Strongyloides has smaller mouth and bigger genital
primordium.
Skill Certification
(05) Scored

writes the report
Score


142
22. Laboratory diagnosis of hepatic
infections
Competencies
 MI3.7: Describe the epidemiology, the etio-pathogenesis and discuss theviral markers in the
evolution of viral hepatitis. Discuss themodalities in the diagnosis and prevention of viral
hepatitis
 MI3.8: Choose the appropriate laboratory test in the diagnosis of viralhepatitis with emphasis
on viral markers

At the end of the teaching-learning session the student shall be able to:
 Enumerate viruses causing hepatitis
 Describe modes of transmission of viruses causing hepatitis
 Describe pathogenesis, clinical features and complications of viruses causing hepatitis
 Describe laboratory diagnosis of viruses causing hepatitis
 Suggest appropriate sample and laboratory diagnostic test for a given case of viral hepatitis
 Interpret the laboratory diagnostic test results of viral hepatitis
 Suggest appropriate measures to prevent viral hepatitis
143
Exercise 22.1:
Clinical case: An outbreak of fever and jaundice
Diagnosis is HAV hepatitis.
1. List other laboratory investigations to diagnose/confirm this condition and expected results
Anti HAV IgM antibody detection by ELISA. HAV antigen detection in stool sample by
ELISA. Detection of HAV particles in stool sample by immunoelectron microscopy.
2. Name other organisms that can produce similar presentation
HEV.
3. Suggest tests and expected results to differentiate other organisms causing similar illness
Anti HAV IgM antibody detection by ELISA indicates acute HAV infection.
4. Explain how other people in the locality could have acquired this infection
Ingestion of water contaminated with feces containing HAV particles excreted by the patient.
5. How is the prognosis of this infection?
Prognosis is excellent.
144
Exercise 22.2:
Clinical case: An intravenous drug abuser with jaundice
Diagnosis is Acute HBV hepatitis

1. List modes of transmission of this infection
Blood and blood products transfusion. Needle prick injuries. Surgery. Dental procedure.
Shared razors and tooth brushes. Sexual transmission particularly in homosexual males.
Mother to fetus, mother to baby during delivery and during breast feeding.
2. Suggest test to know the infectiousness of patient

Detection of HBeAg and HBV DNA indicates active viral replication and patient is highly
infectious.
3. Suggest test to know if the infection is acute or chronic
Detection of Anti HBc antibody IgM indicates acute hepatitis B.
Detection of Anti HBc antibody IgG indicates chronic hepatitis B
4. Suggest measures to prevent this infection
Active immunization by Hepatitis B vaccine.

Passive immunization by HBIG.
Health education. Safe aseptic injection and surgical practices. Safe sex practice.
Screening blood, semen, organs before donation.
145
Exercise 22.3:
Clinical case: A case of fever with jaundice for a few months
Diagnosis is Chronic viral hepatitis B
1. Comment on the stage and infectivity of the infection
Stage of the infection is chronic hepatitis. Presence of HBeAg and HBV DNA indicates patient
is infectious.
2. Enumerate the modes of transmission of this infection
Blood and blood products transfusion. Needle prick injuries. Surgery. Dental
procedure. Shared razors and tooth brushes. Sexual transmission particularly in
homosexual males. Mother to fetus, mother to baby during delivery and during breast
feeding.
3. How is the general prognosis of this condition?
Worse with age
4. Describe briefly the preventive measures of this condition
Active immunization by Hepatitis B vaccine.
Passive immunization by HBIG.
Health education. Safe aseptic injection and surgical practices. Safe sex practice.
Screening blood, semen, organs before donation.
5. Various combinations of serological markers of hepatitis B infection are provided. Interpret
the results and identify the condition
S.No Symptoms HBsAg Anti-HBs Anti-HBc HBeAg Anti-HBe Interpretation
1 Absent + - - - - Early acute

hepatitis B in
incubation
period
2 Present + - IgM + - Acute
hepatitis B.
infectivity
high.
3 Present + - IgG - - Chronic
hepatitis B.
infectivity
low.
4 Absent - + IgG - +/- Recovery
5 Absent - + - - - Post
vaccination
146
Exercise: 22.4
Clinical Case: An asymptomatic person with an abnormal liver seromarker anti HCV IgG
1. What is the clinical diagnosis?

Diagnosis is Hepatitis C Carrier
2. How this person could have acquired the infection?
Blood, blood products, organs transfusion. Needle prick injuries. Surgery. Dental
procedure. Injection drug abusers. Sexual transmission rare. Mother to fetus during
pregnancy.
3. What are the possible outcomes of this condition?

Patient may develop chronic hepatitis if the infection is active.
4. Enumerate other tests that can be done to diagnose/confirm this infection
Detection of HCV RNA by RTPCR indicates active infection.
Exercise 22.5:
Clinical case: A pregnant lady with jaundice
Diagnosis is acute hepatitis E
1. What is the mode of transmission of this infection?

Ingestion of food or water contaminated with HEV.
2. Describe the prognosis of this condition
Pregnant may develop fulminant hepatitis.
147
3. Name other viruses causing hepatitis and their modes of transmission
HAV and HEV are transmitted by feco-oral route.
HBV, HCV, HDV are usually transmitted by percutaneous, sexual and vertical routes.
4. Describe briefly how infection by other hepatic viruses can be ruled out
Absence of antiHAV rules out HAV infection
Absence of HBsAg rules out HBV and HDV infections
Absence of antiHCV IgG rules out HCV infection
Assessment

session
Total score /15
148
Exercise 22.6:
Clinical case: A case of tender hepatomegaly
Diagnosis is amoebic liver abscess
1. Identify the pathogen
Entamoeba histolytica
2. Describe pathogenesis briefly
Liver is the most common site involved in extraintestinal amoebiasis. Most common site
is posterosuperior surface of right lobe. Trophozoites occlude hepatic venules leading to
necrosis of hepatocytes. Anchovy sauce pus formed due to inflammatory response to the
site of infection.
3. Name other parts of the body where such lesions can be found
Lungs, brain, spleen, genitourinary tract.

4. Describe the appropriate sample and its collection procedure to detect the organism
Ultrasound guided pus aspiration.
5. Name the microbiological tests to confirm this infection

Trophozoites detection in pus sample by microscopy. Antigen detection in pus sample by
ELISA. Antibody detection in serum sample by ELISA. Nucleic acid detection in pus sample by
PCR.
149
Exercise 22.7:
Clinical case:A case of cystic hepatic lesion
Diagnosis is hydatid cyst of the liver.
1. Describe the specimen/microscopic features in the smear
Microscopy of hydatid fluid detects brood capsules and protoscolices.

2. Identify the etiological agent
Echinococcus granulosus
3. Describe mode(s) of transmission and source of this infection
Ingestion of food contaminated with dog feces containing Echinococcus eggs.
4. List other laboratory tests to diagnose this infection and the expected findings in each of
them
Histological examination of cyst wall may reveal 3 layers of cyst wall with attached brood
capsules. Antibody detection in serum with ELISA. Water lily sign on ultrasound scan. Detection
of nucleic acid with PCR. Casoni skin test to demonstrate type 1 hypersensitivity reaction to
hydatid fluid.
5. Name other parts of the body where similar lesions can be seen
Lung, kidney, muscle, spleen, soft tissue, brain, bone.
150
Assessment

session
Total score /15
151
BLOCK IV
Skin, Soft Tissue and Musculoskeletal System
Infections
152
23. Laboratory diagnosis of bacterial skin
infections
Competency
MI4.3: Describe the etio-pathogenesis of infections of skin and soft tissueand discuss the clinical
course and the laboratory diagnosis

 Enumerate common manifestations of bacterial skin infections
 Enumerate common bacteria causing skin infections
 Classify common bacteria causing skin infections based on the types of lesions
 Enumerate samples and describe the method of collection of samples to diagnose bacterial
skin infections
 Suggest laboratory diagnostic tests to detect common bacterial skin infections and interpret
the results
 Identify the common bacterial pathogens causing skin infections based on the laboratory
test results provided
 Describe pathogenesis and clinical features of common bacterial skin infections
 Suggest treatment and preventive measures for common bacterial skin infections
 Perform acid fast staining to demonstrate Mycobacterium species in skin samples

Exercise 23.1:
Clinical case:A woman with painful breast swelling
Diagnosis is breast abscess

Breast abscess
2. Suggest appropriate sample to diagnose the condition
Pus sample collected from breast abscess
153
3. Record the findings of the tests displayed and identify the organism
Catalase positive. Slide coagulase test positive. Tube coagulase positive. Golden yellow colonies
on nutrient agar. Beta haemolysis on blood agar. Organism is Staphylococcus aureus.
4. Suggest appropriate antibiotic

Oral therapy with
Dicloxacillin/cephalexin/cefazolin given in MSSA infections.
Clindamycin/linezolid/doxycycline/cotrimoxazole given in MRSA infections.
5. Enumerate the predisposing factors

Breast feeding associated with Poor personal hygiene.
6. Name other common infections caused by this organism

Folliculitis. Furuncle. Impetigo. Carbuncle. Arthritis. Osteomyelitis. Pyomyositis.
Pneumonia. Empyema. Sepsis. Septic shock. Endocarditis. UTI. Pyelonephritis. TSS. Food
poisoning.
7. A heat-fixed smear prepared from the organism isolated from the specimen is provided.
Perform the Gram stain, focus under the microscope, record your observations and interpret the
results
Fig.51.2 A
Observation Gram positive cocci in clusters seen among pink colored pus cells
Inference Staphylococci
154
Examples Staphylococcus aureus
Skill Certification
(05) Scored
Focuses the stained slide appropriately 01
the report
Score


155
Exercise 23.2:
Clinical case:A young man with boils on the leg

Furuncle
2. Suggest appropriate sample to diagnose the condition
Pus sample from the lesion

3. Record the findings of the tests displayed and identify the organism
Catalase positive. Slide coagulase test positive. Tube coagulase positive. Golden yellow colonies
on nutrient agar. Beta haemolysis on blood agar. Organism is Staphylococcus aureus.
4. Suggest appropriate antibiotic

Oral therapy with
Dicloxacillin/cephalexin/cefazolin given in MSSA infections.
Clindamycin/linezolid/doxycycline/cotrimoxazole given in MRSA infections.
5. Enumerate the predisposing factors for such recurrent lesion
Poor personal hygiene. Uncontrolled diabetes mellitus.

Folliculitis. Abscess. Impetigo. Carbuncle. Arthritis. Osteomyelitis. Pyomyositis. Pneumonia.

Empyema. Sepsis. Septic shock. Endocarditis. UTI. Pyelonephritis. TSS. Food poisoning.
156
Exercise 23.3:
Clinical case: A person with discolored limb with bubbles following crush injury
Gas gangrene
2. Suggest appropriate sample to diagnose the condition and briefly describe its
collection and transportation procedure
Necrotic tissue, muscle fragments, exudates from deeper parts of the wound. Specimen to be
transported in RCM broth to the laboratory immediately.
3. Record the findings of the tests displayed and identify the organism(s)
Target haemolysis on blood agar seen. Nagler reaction positive. reverse CAMP test positive.
pathogen is Clostridium perfringens.
4. Suggest other tests to confirm the diagnosis
Heat tolerance test positive. Stormy clot reaction in litmus milk. MALDI-TOF.
5. Suggest appropriate treatment
Early surgical debridement. Combination of penicillin and clindamycin. Hyperbaric oxygen.

Anti alpha toxin antiserum.
157
Exercise 23.4:
Clinical case: A diabetic with painful swelling of leg
Cellulitis by Streptococcus pyogenes

2. Interpret the results and identify the organism
Gram positive cocci in chains. Catalase negative. Pin point colonies with beta haemolysis on
blood agar. Bacitracin sensitive. CAMP test negative. Lancefield grouping.
3. Suggest appropriate antibiotic for this condition
Penicillin. Erythromycin is given in patients allergic to penicillin.
4. Enumerate other common infections caused by this organism
Pharyngitis. Scarlet fever. Pneumonia. Impetigo. Erysipelas. Necrotizing fasciitis.
Myositis. Endocarditis. Osteomyelitis. Puerperal sepsis. Toxic shock syndrome.
5. Name immunological complications that may be produced following infections by

this organism
Acute rheumatic fever. Acute glomerulonephritis. Reactive arthritis.
158
Assessment

session
Total score /15
159
Exercise 23.5:
Clinical case: Wound infection
Diagnosis is wound infection by Escherichia coli.
1. Record and interpret the findings of the tests displayed and identify the organism
Gram negative bacilli on Gram stain of pus sample. LF colonies on MacConkey agar. Indole
production positive. Methyl red reaction positive. VP test negative. Citrate utilization positive.
catalase positive. oxidase negative. Urease negative. Hydrogen sulfide production negative. Gas
production positive. pathogen is Escherichia coli.
2. Name other common organisms causing such infections
Staphylococcus aureus. Escherichia coli. Klebsiella pneuminae. Pseudomonas aeruginosa.

Proteus vulgaris. Enterococcus faecalis. Acinetobacter species.

Diarrhea. UTI. Sepsis. Septic shock. Pneumonia. Meningitis. Peritonitis. Osteomyelitis.
Prostatitis.
4. Suggest an appropriate antibiotic
3rd generation cephalosporins/5 fluoroquinolones tried initially.

5. Briefly describe appropriate sample collection procedure for bacterial culture
Pus/exudates/Wound swab collected.
160
Exercise 23.6:
Clinical case: Burns wound with discharge
Burn wound infection is usually caused by Pseudomonas aeruginosa
1. Record and interpret the findings of the tests displayed and identify the organism
Bluish green pus. Gram negative bacilli on Gram stain of pus sample. Oxidase positive. Diffuse
bluish green pigmentation on nutrient agar. NLF colonies on MacConkey agar. Oxidative in OF
test. Pathogen is Pseudomonas aeruginosa.
2. Name other common organisms causing such infections

Staphylococcus aureus. Enterococcus fecalis. Escherichia coli. Klebsiella pneumoniae.
UTI. Pneumonia. Bacteremia. Sepsis. Septic shock. Endocarditis. Otitis externa. Meningitis.
Arthritis. Osteomyelitis. Corneal ulcers.
4. Suggest an appropriate antibiotic
Antipseudomonal antibiotics like piperacillin, ceftazidime, imipenem, amikacin,

ciprofloxacin used.
161
Assessment

session
Total score /15
162
Exercise 23.7:
Clinical case:A case of hypoasthetic skin patch
1. Identify the clinical condition and justify
Hypopigmented and anesthetic skin lesions are usually seen in tuberculoid leprosy.
2. Describe briefly the sample collection procedure and staining procedure done in this case
Slit skin smear technique followed to collect skin and ear lobe samples.
Nasal blow and nasal scrapings done to collect nasal specimens.
Acid fast stain by Ziehl-Neelsen method is done to see lepra bacilli.
3. Record your observations, draw a diagram and interpret the results
Red colored acid fast bacilli seen in groups and singles. Globi are seen.
Fig.54.3
4. Describe briefly the immunological method to assess the prognosis of this disease
Lepromin test is done with lepra antigen. It demonstrates delayed hypersensitivity reaction to
lepra antigen and indicates an intact host’s CMI response to leprosy. It is used to assess
prognosis of the disease. It is positive in tuberculoid leprosy and negative in lepromatous
leprosy.
163
Exercise 23.8:
An 8 year-old child presented with poor appetite, failure to thrive, on-and-off fever since 1
month. On examination cervical lymph nodes were enlarged, matted and nontender. The
overlying skin appeared normal. A heat-fixed smear prepared from the lymphnode aspirate is
provided. Perform the acid fast stain, focus under the microscope, record your observations and
interpret the results. Diagnosis is Tuberculosis of cervical lymph nodes.
Fig.63.3A
Observations Long, slender and beaded red colored bacilli seen among blue colored pus
cells.
Inference Acid fast bacilli
Examples Mycobacterium tuberculosis
164
Skill Certification
(05) Scored
the report
Score


165
24. Laboratory diagnosis of fungal and viral
skin infections
Competency
MI4.3: Describe the etio-pathogenesis of infections of skin and soft tissueand discuss the clinical
course and the laboratory diagnosis

 Enumerate common manifestations of viral skin infections

 Enumerate common viruses causing skin infections
 Classify common viruses causing skin infections based on the types of lesions
 Enumerate samples and describe the method of collection of samples to diagnose viral skin
infections
 Suggest laboratory diagnostic tests to detect common viral skin infections and interpret the
results
 Identify the common viral pathogens causing skin infections based on the laboratory test
results provided
 Describe pathogenesis and clinical features of common viral skin infections
 Suggest treatment and preventive measures for common viral skin infections
 Enumerate common manifestations of fungal skin infections
 Enumerate common fungi causing skin infections
 Classify common fungi causing skin infections based on the types of lesions
 Enumerate samples and describe the method of collection of samples to diagnose fungal
skin infections
 Suggest laboratory diagnostic tests to detect common fungal skin infections and interpret
the results
 Identify the common fungal pathogens causing skin infections based on the laboratory test
results provided
 Describe pathogenesis and clinical features of common fungal skin infections
 Suggest treatment and preventive measures for common fungal skin infections
166
167
Exercise 24.1:
Clinical case:A case of painful perioral lesions with fever
1. Name the clinical diagnosis and the causative agent
Herpes labialis. It is caused by Herpes simplex virus type 1.

2. Name the probable sample used for preparing the smear
Scrapings obtained from the base of the lesions. Tzanck smear is made with Giemsa stain.
3. Record your observations of microscopy of the lesions and interpret the result
Multinucleated giant cells seen in Tzanck smear.

4. What is the mode of spread of this infection and reason for recurrence
Oropharyngeal contact with infected saliva or direct skin contact. Recurrence is due to
reactivation of latent infection.
5. Name other investigations to diagnose this condition
Virus isolation on conventional cell culture or shell culture. Viral antigen detection by IFA.
Virus specific antibody detection by ELISA. HSV DNA detection by PCR.
6. Enumerate complications of this infection
Encephalitis. Meningitis. Facial palsy. Corneal ulcer. Pneumonitis. Hepatitis.
168
Exercise 24.2:
Clinical case: A case of generalized exanthematous skin lesions and fever
1. Identify the most probable clinical condition and the causative agent
Measles. Pathogen is Measles virus.

2. Suggest laboratory investigations to diagnose the infection
Nasopharyngeal swab collected. Viral antigen detection by ELISA. Viral RNA by RTPCR. Viral
isolation in cell culture. Antibody detection in serum by ELISA
3. Enumerate viruses causing exanthematous lesions. How are they differentiated from this
infection
Exanthems are also caused by Rubella virus, Dengue virus, Coxsackie virus, herpes simplex
virus, VZ virus. Koplik’s spots are pathognomonic to Measles. Rashes typically appears first
behind the ears.
4. Enumerate complications of this infection
Secondary bacterial infections. Pneumonitis. Croup. Diarrhea. SSPE.

5. Suggest prevention of this infection
Treat by Isolation of patient in negative pressure room. Use PPEs and N95 respirator while
treating patients. MMR vaccine in infants.
169
Exercise 24.3
Clinical case: A case of multiple umbilicated papular lesions
1. Identify the clinical condition and the causative agent

Molluscum contagiosum. Pathogen is Molluscum contagiosum virus.
2. Suggest laboratory investigations to diagnose this condition
PCR. Molluscum bodies seen in skin scrapings with histopathology stains.
3. Describe modes of transmission of this infection

Contact with the lesions. Sexual transmission. Fomites.
Assessment

session
Total score /15
170
Exercise 24.4
Clinical case:A case of itchy ring-shaped skin lesions
1. Name the clinical condition
Tinea/ring worm infection.

2. Study the tests displayed test results, record the observations and identify the organism
Fig. 58.3 C and F.

Trichophyton mentagrophytes
3. Describe the procedure of collection and transportation of appropriate sample for the above
test
Skin scrapings collected from the margins of the lesion. Examined microscopically after treating
with 10% KOH.
4. Name other organisms causing similar lesions
Other dermatophytes like Microsporum species and Epidermophyton species can cause ring
worm lesions.
5. Describe mode of transmission
Direct contact with soil/humans/animals infected with fungal spores.
171
Exercise 24.5:
Clinical case:A case of swollen foot with multiple discharging openings
1. Identify the clinical condition and Justify your answer
Mycetoma foot. Swelling on foot with multiple discharging sinuses is characteristic of

Mycetoma.
2. Enumerate organisms causing such lesions
Caused by fungi like Madurella mycetomatis, Aspergillus nidulans, Curvularia species and
bacteria like Actinomadura madurae, Nocardia species, Streptomyces somaliensis.
3. Describe sample collection procedure

Lesion cleaned with antiseptics. Grains are collected on sterile gauze by pressing the sinuses
from periphery.
4. Suggest treatment
Surgical removal of the lesion. It is followed by antifungal agents like itraconazole for
eumycetoma and antibiotics like amikacin plus cotrimoxazole for actinomycetoma.
172
Exercise: 24.6
Clinical case: A diabetic with blackish skin lesions on face
1. Identify the causative agent and Justify your answer

Rhino-orbital mucormycosis. Pathogen is Rhizopus. Diabetes is the most important risk factor.
2. Name the sample used for doing the tests displayed

Tissue biopsy.
3. Draw neat labeled diagram of microscopic appearance of the organism
Fig.69.5A
4. Suggest a rapid and simple test for preliminary identification of the organism
Histopathology of tissue section shows broad, aseptate, hyaline hyphae.
5. Name other organisms causing similar lesions
Mucor, Rhizomucor, Lichtheimia can also mucormycosis.
6. Enumerate predisposing factors for this condition
Diabetic ketoacidosis. Iron therapy. End stage renal disease. Steroid therapy. Neutropenia.
7. Suggest appropriate treatmentfor this condition
Amphotericin B deoxycholate is the drug of choice. Posaconazole is the alternative drug.
173
Exercise 24.7:
Clinical case: A cancer patient with white oral lesions

Oral thrush/candidiasis
2. Identify the organism and Justify your answer
Fig.38.1A
Candidiasis is common in cancer patients due to low immunity.
3. Name common species of this organism and describe how the most common pathogenic
species is identified
C.albicans. C.tropicalis. C.glabrata. C.krusei.
C.albicans is differentiated by positive Germ tube test.
4. Name other lesions caused by this organism
Vulvovaginitis. Onychomycosis. Septicemia. Keratoconjunctivitis. UTI. Pulmonary
infection.
5. Enumerate predisposing factors for this condition
Diabetes mellitus, malignancy, AIDS, immunosuppressive treatment.
174
Assessment

session
Total score /15
175
25. Laboratory diagnosis of
Musculoskeletalinfections
Competency
MI 4.2. Describe the etiopathogenesis, clinical course and discuss thelaboratory diagnosis of
bone & joint infections

At the end of the teaching-learning session the student shall be able to:
 MI 4.2.1. Classify osteomyelitis based on the duration
 MI 4.2.2. Enumerate organisms causing osteomyelitis based on the duration of the infection
 MI 4.2.3. Describe pathogenesis and clinical features of acute and chronic osteomyelitis
 MI 4.2.4. Suggest appropriate samples to be collected and laboratory tests to be performed
for acute and chronic osteomyelitis
 MI 4.2.5. Interpret the laboratory tests performed for diagnosis of acute and chronic
osteomyelitis to identify the etiological agent and choose appropriate treatment
 MI 4.2.6. Enumerate organisms causing arthritis
 MI 4.2.7. Describe pathogenesis and clinical features of septic arthritis
 MI 4.2.8. . Suggest appropriate samples to be collected and laboratory tests to be performed
for septic arthritis
 MI 4.2.9. Interpret the laboratory tests performed for diagnosis of septic arthritis to identify
the etiological agent and choose appropriate treatment
176
Exercise25.1:
Clinical case: A case of painful swollen joint with fever
Diagnosis is septic arthritis.
1. Record the findings and interpret the results

Gram positive cocci in clusters in Gram stain. Catalase positive. Golden yellow colonies on
nutrient agar. Beta haemolysis on blood agar.
Fermentor in OF test. Slide coagulase test positive. Tube coagulase test positive.
Staphylococcus aureus.
3. Enumerate other supportive microbiological tests useful in this condition
Protein A detection test positive. Growth on selective media like mannitol salt agar positive.
DNAse test positive.
4. Describe the pathogenesis briefly
Pathogen gains entry into the joint either through blood supply or direct invasion. Then it
establishes by evading local host defense mechanisms. Local inflammatory response to
infection leads to arthritis.
5.The patient is being administered ceftriaxone. Comment

Resistance is common to ceftriaxone. Therefore, Antimicrobial susceptibility test
should be done to know the antibiotics to which pathogen is sensitive and
prescribed accordingly.
177
Exercise 25.2:
Clinical case: A case of painful swollen joint and skin rashes.
Patient has promiscuous behavior.
Gonococcal septic arthritis

Neisseria gonorrhoeae
2. Describe pathogenesis briefly

Polyarthritis occurs after disseminated gonococcal bacteremia. It is a complication
that may follow gonococcal urethritis.
3. Name other tests to confirm the diagnosis and expected results

Blood/synovial fluid culture using MALDI-TOF.
Nucleic acid detection in synovial fluid by PCR.
3rd generation cephalosporins like Ceftriaxone/cefixime.
Exercise 25.3:
Clinical case: A case of painful swollen limb with fever after trauma
1. Diagnose the clinical condition
Acute osteomyelitis
2. Identify the causative agent
Gram positive cocci in clusters among pus cells. Catalase positive. Coagulase positive. Pathogen
is Staphylococcus aureus.
178
3. List the predisposing factors for development of this condition
Trauma predisposes to invasion of the local commensals like S.aureus.
S.aureus is known to exhibit resistance to multiple antibiotics. Therefore,
Antibiotics given as per the antimicrobial susceptibility report.
Assessment

session
Total score /15
179
Exercise 25.4:
Clinical case: An adult with chronic backache and weight loss
1. Diagnose the clinical condition and justify
Diagnosis is chronic vertebral osteomyelitis due to TB. Chronic nature associated

with weight loss are suggestive of TB spine.
2. Perform Ziehl-Neelsen staining on the given heat-fixed smear. Focus the smear under
microscope, record your observations and interpret the results
Fig.63.3A
Observations Long, slender, beaded red colored bacilli seen
Example Mycobacterium tuberculosis
180
3.Enumerate other tests to diagnose/confirm this condition along with the expected
findings
Culture, PCR of the aspirated material.

4.Suggest investigations to choose most appropriate treatment in this case
Abdomen X ray and MRI scan.
Skill Certification
(05) Scored
the report
Score


181
BLOCK V
Central nervous system infections
182
26.Laboratory diagnosis of Meningitis
Competency
MI 5.3 Identify the microbial agents causing meningitis
At the end of this practical, the students will be able to:

 Enlist pathogens causing infections in Central Nervous System.
 Enlist the appropriate sample to be collected in case of CNS infection
 Describe collection, transport and storage of CSF.
 Identify the microbial agents causing meningitis based upon the macroscopic and
microscopic findings of CSF.
 Choose the appropriate laboratory investigations to be performed for diagnosis of CNS
infections.
 Interpret the results of the laboratory tests performed in diagnosis of the CNS infections.
183
Exercise 26.1:
Clinical Case: A febrile drowsy and irritable child with stiff neck. No bacteria are seen in
Gram stain
1. What is the most likely clinical diagnosis in this child? Give reasons
Asceptic meningitis. No bacteria seen in Gram stain rules out bacterial meningitis.
Viruses are next possible cause of meningitis in children.
2. Suggest investigations to be carried out to confirm the diagnosis
CSF analysis. PCR. viral culture. Antibody detection. Antigen detection.
3. Keeping the age of patient, enlist probable pathogens in the order of priority.
Enteroviruses. HSV 1. Arboviruses.
4. Describe the precautions to be taken while collection and transportation of CSF.
CSF collected by lumbar puncture under strict asceptic conditions. It should be send immediately to
the laboratory. It should be processed immediately and never be refrigerated.
5. Describe microbiological investigations for diagnosis of meningitis.
PCR. viral culture. Antibody detection. Antigen detection.
184
Exercise 26.2:
Clinical Case: An AIDS patient with fever, headache and stiff neck
1. What is the most probable diagnosis, Justify your answer
Cryptococcal meningitis. Cryptococcus neoformans is a common opportunistic

fungal pathogen that causes meningitis in AIDS patients.
2. Draw a labeled diagram of this organism as seen in Indian ink preparation
Fig.75.9 A
3. Describe other microbiological investigations that can be done to diagnose this disease.
Capsule antigen detection in CSF by latex agglutination test. CSF culture.
4. Name the media that will be used for isolation of the organism.
Niger seed agar and bird seed agar.
185
Exercise 26.3:
Clinical Case: A case of fever, headache and stiff neck. Gram negative diplococcic seen in Gram
stain.
Diagnosis is pyogenic meningitis.
1. Name the possible pathogen

Neisseria meningitidis
2. Name the media that will be used for isolation of the organism.
Blood agar
3. Enlist the types of infection seen in CNS with their key etiological agents
And mention the differences between CSF findings in various types of CNS infections
Bacterial Viral infection Fungal Parasitic
infections infection infection
Gross examination Purulent clear - -
Microscopic findings Bacteria Viral particles Fungal Parasitic forms
seen not seen under elements seen seen
normal
microscopy
Biochemical findings Glucose Glucose - -
decreased normal
Cell count and Neutrophil Lymphocyte - -
morphology count raised count raised
Any other point Protein Protein normal - -
raised
186
4. From another patient with similar complaints CSF culture yielded growth. A heat-fixed
smear prepared from the colonies grown from the specimen is provided. Perform the
Gram stain, focus under the microscope, record your observations and interpret the
results
Fig.71.2
Observations Capsulated Gram negative
half moon shaped diplococci
seen
Inference Meningococci
Example Neisseria meningitidis
187
Skill Assessment
(05) Scored

writes the report
Score


188
27. Laboratory diagnosis of Encephalitis
Competency
MI5.2: Describe the etiopathogenesis, clinical course and discuss the laboratory diagnosis of
encephalitis

 Enlist pathogens causing encephalitis
 Enlist the appropriate sample to be collected in case of encephalitis
 Choose the appropriate laboratory investigations to be performed for diagnosis of
encephalitis
Exercise27.1:
Clinical case: A case of fever and seizure
Encephalitis
1) Suggest investigations that need to be done to confirm the probable

CSF analysis. Antigen detection in CSF. Antibody detection in serum. Nucleic acid detection in CSF by
PCR.
2) How do you differentiate a case of encephalitis from meningitis?
Fever with Signs of meningism like Neck stiffness, Kernig’s sign, Brudzinski’s sign are seen in
meningitis.
Fever with Loss of consciousness, seizures, raised intracranial pressure are seen in encephalitis.
3) Enlist the infective causes of encephalitis and their supportive laboratory findings.
HSV and Nipah viral encephalitis can be diagnosed by CSF PCR.
IgM capture antibody ELISA is used to detect Japanese encephalitis, West Nile encephalitis.
Direct immunofluorescence test on hair follicle of the nape of the neck is used to detect Rabies.
189
Assessment

session
Total score /15
190
BLOCK VI
Respiratory tract infections
191
28. Laboratory diagnosis of Upper
respiratory tract infections (URTI)
Competencies
MI 6.2 Identify the common etiologic agents of upper respiratory tract infections (Gram Stain)
 Enlist pathogens causing infections in respiratory system.

 Describe collection, transport and storage of various respiratory specimens.
 Identify the common etiologic agents of upper respiratory tract infections from Gram stain
smear.
 Choose the appropriate microbiological investigation to be performed in a given case of
URTI.
 Interpret the results of the laboratory tests used in diagnosis of URTI.
 Demonstrations of upper respiratory pathogens like Corynebacterium diphtheria (in Gram
stain, Albert stain), Staphylococcus aureus, Streptococcus pyogenes
192
Exercise 28.1:
Clinical Case: A febrile child with tough leathery greyishwhite membrane in throat
1) What is the clinical diagnosis? Justify your answer

Diphtheria. Tough, grayish white, leathery membrane on tonsils in children is usually suggestive
of diphtheria.
2) Suggest appropriate sample and briefly describe the sample collection procedure to diagnose
this infection
Throat swab and a piece of membrane.
3) A smear of the sample stained with a special stain is focused. Record your observations and
interpret the results to identify the pathogen in this case. Fig. 60.2C
Observation Green bacilli with dark blue granules at both the ends.
Inference Corynebacterium diphtheriae
4) Suggest other laboratory tests to confirm the diagnosis
Conventional culture and biochemical tests or automated systems like MALDI-TOF/VITEK are
used to confirm the diagnosis. Toxin detection by ICT/ELISA is used to confirm toxigenic nature
of the pathogen.
193
5) Describe prevention of this condition
Diphtheria vaccine is given under NIS to protect children from diphtheria. Household contacts of
the infected child are given a booster dose of the vaccine along with penicillin G or
erythromycin.
Exercise 28.2:
Heat-fixed smear of the colonies obtained on blood agar from the sample obtained from the
above case is provided. Perform the Gram stain, focus under the microscope, record your
observations and interpret the results Fig. 60.2B
Observations Pairs of Gram positive bacilli arranged in V shape, L shape and parallel.
It is described as cuneiform arrangement.
Inference Diphtheria bacilli
Example Corynebacterium diphtheriae
194
Skill Certification
(05) Scored
the report
Score


195
29. Laboratory diagnosis of Lower
respiratory tract infections (LRTI)
Competencies
MI 6.3: Identify the common etiologic agents of lower respiratory tract infections (Including
CoVID-19) (Gram Stain & Acid Fast Stain) Grams Stain-5, Z-N stain-3, hand hygiene, PPE-3
 Enlist pathogens causing infections in Respiratory System.

 Describe collection, transport and storage of various respiratory specimens.
 Identify the common etiologic agents of lower respiratory tract infections from Gram Stain &
Acid fast stained smear.
 Choose the appropriate microbiological investigation to be performed in a given case of
LRTI.
 Interpret the results of the laboratory tests used in diagnosis of LRTI.
 Demonstrations of respiratory pathogens like Streptococcus pneumoniae, Klebsiella
pneumoniae (in Gram stain, colony characteristics, key identification test),
Mycobacterium tuberuculosis (in ZN stain, Growth on LJ media)
Exercise 29.1:
Clinical Case: A case of acute fever and cough with purulent expectoration and chest pain.
(a) Identify the bacterial morphology seen in the Gram stain.
Gram positive cocci in pairs enclosed in a capsule.
(b) Name the most likely infective cause of the case, Justify your answer
Streptococcus pneumoniae. It is the leading cause of bacterial lobar pneumonia.
196
_______________________________________________________________________
(c) Describe other microbiological investigations that can be done to confirm the diagnosis of
this case.
Culture on blood agar. India ink stain to demonstrate capsule. Biochemical tests like bile
solubility, inulin fermentation, optochin sensitivity. Automated methods like
MALDI-TOF/VITEK. PCR.
Assessment

session
Total score /15
197
Exercise 29.2:
Clinical Case: A case of longstanding productive cough with weight loss
1. Write the most likely diagnosis
Pulmonary tuberculosis
2. Explain the microbiological investigations that can help you in confirming the
laboratory diagnosis of the disease
Sputum sample collected. Acid fast stain, conventional culture, BACTEC MGIT, nucleid
acid detection by PCR.
3. A heat-fixed smear prepared from the sputum sample of the patient is provided. Perform the
suitable staining procedure, focus under the microscope, record your observations and interpret
the results
Fig.63.A
Observation Long, slender, beded red colored bacilli seen among blue colored
pus cells and epithelial cells
Example Mycobacterium tuberculosis
198
Skill Certification
Competency No.: MI 1.2 Competency: Ziehl-Neelsen stain (5)
(05) Scored
the report
Score


199
Exercise 29.3:
Clinical case: A case of old treated tuberculosis presenting with hemoptysis
1) What is the clinical diagnosis?

Aspergilloma
2) Record the observations based on the growth and microscopy demonstrated. Draw a neat-
labeled diagram of the organism and identify.
Fig.69.7A
Aspergillus fumigates accounts for most of the cases of acute pulmonary aspergillosis.
200
3) Differentiate other common species of this organism
Aspergillus fumigatus Aspergillus flavus Aspergillus niger
Colonies are green and Colonies are yellow and Colonies are black and
velvety. velvety. powdery.
Conidia arise from upper third Conidia arise from upper two Conidia arise from entire
of the vesicle. third of the vesicle. vesicle.
4) Enumerate other common infections caused by this organism

ABPA. Asthma. Angioinvasive pulmonary aspergillosis. Sinusitis. Endocarditis. Keratitis. Otitis
externa. Onychomycosis. Cutaneous aspergillosis. Haemorrhagic infarction. Mycotoxicosis.
5) Enumerate predisposing factors for developing such infections
Glucocorticoid use. Neutropenia. Neutrophil dysfunction. Underlying TB or COPD or
pneumonia.
201
Exercise 29.4:
You are about to provide care to a patient diagnosed with Covid-19 admitted in an Intensive Care
Unit. Choose appropriate PPE required and demonstrate donning and doffing of the PPE.
Perform hand hygiene wherever appropriate.
Hand hygiene

(Score=0)
1 Removes all hand accessories ( finger ring wrist 1.0
watch. etc.) (0.5) &Applies sufficient amount of
soap/hand wash /hand rub (0.5)
3 Rubs back of palm on both sides (0.5 + 0.5) 1.0
4 Follows rotational rubbing of thumb on both sides 1.0
(0.5 + 0.5)
5 Rubs back of fingers on palm on both sides (0.5 +0.5) 1.0
7 Rubs nails on palms on both sides (0.5 + 0.5) 1.0
Total score 10 /10
202

(Score=0)
Donning Glove:1 Wears by touching and pulling only the 1.0
edge of the cuff
Glove:2 a) Wears by pulling the external surface 1.0

of second glove by
Doffing Glove:1 Removes first glove by using the other 1.0

gloved hand, grasps the palm area of the
first glove & peels it off.
Glove:2 a) Holds the removed glove in the other 1.0

gloved hand (0.5) and
b) slides fingers of the ungloved hand
under the other glove at wrist and peels
off second glove over first glove. (0.5)

b) Performs hand hygiene after gloves removal
(0.5)
Total score 5.0 /05
203
Sequential Donning (wearing) of PPE

(Score=0)
Performs proper hand hygiene 1.0
Wears the gown first in sequence 0.25
Wears the gown by fully covering torso from neck to 0.5

knees, arms to end of wrists, and wraps around the back
and fasten it at the waist and back of neck
Wears the mask second in sequence 0.25
Pulls the straps tight and pulls the mask to below chin and 0.5
then applies knots.
Presses on the nasal bridge part of the mask to seal tightly 0.5
and for N95 respirator, performs fit check.
Wears the goggles/face shield third in sequence 0.25
Wears the goggles/face shield by touching the front part 0.5

only
Wears the gloves last in sequence 0.25
Glove:1 Wears by touching and pulling only the edge of 0.5

the cuff

second glove bythe finger of gloved hand (0.25)
Total score 5.0 /05
204
Sequential Doffing (removal) of PPE
(Score=0)
Removes the gloves first 0.5
Does not touch outside of the gloves (contaminated) 0.5
Glove:1 Removes first glove by using the other gloved hand, 0.5
grasps the palm area of the first glove & peels it off.
Glove:2 a) Holds the removed glove in the other gloved hand 0.5
(0.25) and
b) slides fingers of the ungloved hand under the other
glove at wrist and peels off second glove over first
glove. (0.25)
Discards the gloves into red bin 0.5
Performs hand hygiene after gloves removal 0.5
Removes face shield/ goggles second in sequence 0.5
Removes face shield/ goggles by touching the sides only and 0.5
bending forward
Discards face shield/ goggles into red bin 0.5
Does not touch the front part of the gown while removing 0.5
Unfastens the gown ties, taking care that sleeves do not touch the 0.5
body while reaching for ties
Pulls the gown away from neck and shoulders, by touching inside 0.5
of gown only
Turn the gown inside out and rolls it into a bundle and discards 0.5
Discards the gown into yellowbin 0.5
Performs hand hygiene after removal 0.5
Takes off mask fourth in sequence 0.5
Does not touch front part of the mask. 0.5
Unties the lower knot first, then the upper knot and removes the
mask by holding its straps, without touching the front
Discards the mask into yellowbin 0.5
Performs hand hygiene at the last after removal of all PPE 0.5
Total score 10 /10
205
BLOCK VII
Genitourinary & Sexually transmitted
infections
206
30. Laboratory diagnosis of Genitourinary
and Sexually transmitted diseases
(Urethritis, Genital ulcers)
Competency
 MI 7.1 Describe the etio-pathogenesis and discuss the laboratory diagnosis of infections of
genitourinary system
 MI 7.2 Describe the etio-pathogenesis and discuss the laboratory diagnosis of sexually
transmitted infections. Recommend preventive measures
Specific Learning objectives :
 Enlist various sexually transmitted pathogens.

 Describe collection, transport and storage of various specimens for diagnosis of
genitourinary &sexually transmitted infections.
 Choose the appropriate microbiological investigation to be conducted in a case of genital
tract / sexually transmitted infection.
 Interpret the results of the microbiological investigation conducted in a case of genital tract /
sexually transmitted infection.
 Counsel a patient of genital tract/ sexually transmitted infection about preventive aspects of
such infections.
207
Exercise 30.1:
Clinical Case: A case of painless genital ulcer
1) What is the most likely clinical diagnosis of the case?

Primary syphilis,
2. Describe the sample collection and laboratory techniques that can be used in the
diagnosis of the disease.
A drop of exudates from the ulcer is collected. Direct microscopy is done with dark ground
microscope or direct fluorescence antibody staining or silver impregnation method to
demonstrate treponemes. Antibody detection is done with non specific test like VDRL/RPR
test and confirmed by specific tests such as FTA-ABS.
3. Draw the microscopic examination findings from the sample collected from ulcer.
fig. 77.3A
208
Exercise 30.2:
Clinical Case: A case of painful genital lesions
1. What is the most probable clinical diagnosis of the case?
Herpes genitalis
2. Describe the sample collections required for diagnosis of this disease.
Scrapings from the base of the lesions are collected on a glass slide and Giemsa stain
done. This is called as Tzanck smear.
3. Describe the microscopic examination that can be performed and draw the diagram.
fig. 56.3A
4. Briefly describe other diagnostic tests for this infection
Virus isolation in cell culture. Viral antigen detection by IFA. Nucleic acid detection by PCR.
Antibody detection in serum by ELISA.
209
5. Explain the reason for recurrence of this infection
Reactivation of the latent virus
6. Enumerate complications of this condition
Inguinal lymphadenopathy. Urethritis. Vulvovaginitis. Cervicitis. Endometritis. Salpingitis.
Exercise 30.3:
Clinical Case: A woman with vaginal itching and discharge
1. Name the most likely organism seen in the wet preparations.
Trichomonas vaginalis
2. Describe the laboratory diagnosis of this disease.

Vaginal discharge is collected. Wet mount or DFA test to demonstrate parasite. Culture.
Antigen detection by ELISA. Nucleic acid detection by PCR.
3. Name other organisms transmitted in the similar manner

Candida albicans. Treponema pallidum. Neisseria gonorrhoeae. Haemophilus ducreyi. Herpes
simplex virus. Chlamydia trachomatis. HPV. HIV. HBV. HCV.
Metronidazole is the drug of choice. Both sexual partners should be treated simultaneously.
210
Assessment

session
Total score /15
211
31. Laboratory diagnosis of Urinary tract
infections (UTI)
Competency
MI 7.3: Describe the etio-pathogenesis, clinical features, the appropriate method for specimen
collection, and discuss the laboratory diagnosis of Urinary tract infections

 Enlist pathogens causing infections in Urinary system.
 Describe collection, transport and storage of urine for diagnosis of urinary tract infections.
 Give instructions to patients for mid stream sample collection and transportation.
 Choose the appropriate sample to be collected in a case of UTI (upper/lower UTI)
 Interpret the results of the laboratory tests used in diagnosis of the UTI.
Exercise 31.1:
Clinical Case: A case of fever and burning micturition
Diagnosis is UTI
1. Identify the culture media, culture technique and purpose of the performing this in diagnosis of
urinary tract infections.
MSU sample is collected. Nutrient agar, blood agar, MacConkey agar, CLED agar are used to
isolate bacterial pathogens that cause UTI. Quantitative culture using a standardized loop or
pour plate method is done to estimate significant bacteriuria.
2. Which organisms are probably responsible for the woman’s infection?
Escherichia coli. Klebsiella pneumoniae. Pseudomonas aeruginosa. Staphylococcus
aureus. Enterococcus fecalis. Proteus vulgaris. Candida albicans. Staphylococcus
saprophyticus.
212
3. What is the significance of bacterial count in the report of urine?
Presence of significant bacteriuria indicates infection is likely. This helps in differentiating
from contamination of the urine sample.
4. Write instructions that must have been given to her for appropriate sample collection and
transportation.
Clean properly the urethral meatus and MSU collected in a wide mouthed screw
capped container. Sample is immediately transported to the laboratory fro processing.
Exercise 31.2:A young newly married female presented to medicine OPD with increased
frequency and painful urination. She does not have any other complains like fever. Name the
possible pathogens in her case.
Staphylococcus saprophyticus.
Exercise 31.3:A pregnant female was screened during her routine antenatal checkup. Her urine
culture grew Escherichia coli with significant count. Elaborate on the way this report needs to be
interpreted and impact of this on the outcome of pregnancy.
Asymptomatic bacteriuria is clinically significant in pregnancy. It should be
treated as the risk of complications to mother and fetus are high.
Exercise 31.4:Urine culture of a catheterized adult male with stroke reported growth of two
organisms with significant count but patient is not having any symptoms. Write about your
follow up plan for this case.
Asymptomatic bacteriuria is not clinically significant in this patient.
213
Assessment

session
Total score /15
214
ANNEXURE
GUIDE TO TEACHERS
FOR CONDUCTING PRACTICAL
TEACHING-LEARNING SESSIONS&
ASSESSMENT
Dr. JyotiNagmoti
Dr. Apurba S Sastry
215
Dr. SumanP Singh
Dr. Anand B Janagond
Suggestions for the conduct of Teaching

Learning and Assessment Activities
1. This section provides appropriate interactive teaching learning methods (small group
discussion, demonstrations, DOAP, role plays etc.), time slots and key words for creating
interesting case scenarios to ensure enhanced skill learning.
2. The purpose of this book is to record all the teaching-learning-assessment events of the
student, which include
- Practical exercises of specified competencies
- Certifiable skills
- AETCOM skills
- Assessment of day-to-day performance and skill certification
- Feedback provided by the teacher
3. These entries are evidences for the work done by the student.
4. The faculty shall ascertain the student’s learning by regular assessment. If the
performance of the student is of a level below expectation, remedial measures shall be
suggested by the faculty
5. It is the responsibility of the student to bring this record book to every practical class,
record the relevant tasks performed/discussed and show it to the faculty assigned for
assessment, feedback and signature regularly
6. This practical record will reflect the progress of the student’s acquisition of knowledge
and skills pertaining to the competencies specified, including the certifiable skills. This is
also an evidence for the entries made in the logbook.
216
7. At the end of the academic year the student’s record will be certified by the Faculty
in-charge and the Head of the Department after ensuring that the progress is at the
expected level or above
217
Table of Contents
I General Microbiology, Immunology & Hospital Infection Control
1 A-Introduction to Microbiology laboratory MI 1.1
B - Microscopy MI 1.2
2 1. General principles of Laboratory diagnosis of MI 8.9,
Bacterial diseases.(Sample collection, transportation, MI 8.10
bacterial identification methods in general) MI 8.7
2. Demonstration of respect for patient samples sent MI 8.11
for laboratory investigations**
3. Hand hygiene, PPE- Donning & Doffing of hand MI8.7
gloves (1)*
3 1. Direct methods of bacterial detection MI 1.2
c) Simple staining
d) Hanging drop
2. Gram staining (1)* MI1.2
4 Ziehl-Neelsen staining (1)* MI 1.2
5 Bacterial culture: Culture media, methods and MI 1.1
identification techniques (conventional and automated)
6 Antibiotic sensitivity testing MI 1.6
7 Indirect methods of infectious disease diagnosis MI 1.8, MI
(Immunological diagnostic tests) 8.15
8 General principles of laboratory diagnosis of Viral MI 1.1
diseases
9 1. General principles of laboratory diagnosis of MI 1.2
Parasitic diseases
2. Stool Examination (1)* MI1.2
10 General principles of laboratory diagnosis of Fungal MI 1.1
diseases
11 Hospital infection control – I: Hand hygiene, PPE MI8.5, MI8.6,
(Donning & Doffing) MI8.7, MI8.8
Hand hygiene, PPE (2)* MI8.7
12 Hospital infection control - II: Sterilization & MI8.5, MI8.6,
Disinfection, Biomedical waste management, Needle MI8.7, MI8.8
stick injuries
218
13 Laboratory diagnosis of Rheumatic heart disease, MI2.1, MI2.2,
Infective endocarditis and sepsis MI2.3
Gram Stain (2)* MI1.2
14 Laboratory diagnosis of Brucellosis, Leptospirosis, MI4.3, MI8.1
Dengue fever, Scrub typhus, Candidemia
15 Laboratory diagnosis of Enteric fever MI3.3, MI3.4
16 Laboratory diagnosis of Malaria MI2.5, MI2.6
17 Laboratory diagnosis of Filariasis and Leishmaniasis MI2.5, MI2.6
18 1. Laboratory diagnosis of HIV infection MI2.7
2. Confidentiality pertaining to patient's identity in MI8.12,
laboratory result** MI8.14
19 Laboratory diagnosis of Diarrhea MI1.2, MI3.1,
MI3.2
Ziehl-Neelsen staining (2)* MI1.2
Stool Examination (2)* MI1.2
20 Laboratory diagnosis of Dysentery MI1.2, MI3.1,
MI3.2
Stool Examination (3)
21 Laboratory Diagnosis of Intestinal helminthic MI1.2, MI2.4,
infections MI2.5, MI3.1,
MI3.2
Stool Examination (4)* MI1.2
22 Laboratory diagnosis of Hepatic infections MI3.7, MI3.8
23 Laboratory diagnosis of skin infections-I MI4.3
(Bacterial: Furuncle, cellulitis, Surgical site infection,
Burn wound infection, Leprosy)
Gram Stain (3)* MI1.2
Ziehl-Neelsen staining (3)* MI1.2
24 Laboratory diagnosis of skin infections-II MI4.3
(Fungal and Viral infections)
25 Laboratory diagnosis of musculoskeletal infections MI4.2
(Arthritis, Osteomyelitis) MI1.2
Ziehl-Neelsen staining (4)*
219
26 Laboratory diagnosis of Meningitis (Pyogenic, MI5.3
Tubercular, Cryptococcal& Aseptic)
* Gram Stain (4) MI1.2
27 Laboratory diagnosis of Encephalitis MI5.3
28 Laboratory diagnosis of Upper respiratory tract MI6.2
infections
Gram Stain(5)* MI1.2, MI6.2
29 Laboratory diagnosis of Lower respiratory tract MI6.2, MI6.3
infections
Ziehl-Neelsen stain (5)* MI1.2, MI6.3
Hand hygiene, PPE (3)* MI8.7
30 Laboratory diagnosis of Urinary tract infections MI7.1, MI7.2
31 Laboratory diagnosis of Genitourinary and Sexually MI7.3
transmitted diseases (Urethritis, Genital ulcers)
220
Time-Line for Teaching-Learning Sessions
and Assessment
S.No Topics, Exercises and Assessment 2.00 Hours
I General Microbiology, Immunology & Hospital Infection Control, SLOTS
1.A Introduction to Microbiology laboratory
1.B Microscopy 1
Assessment
General principles of Laboratory diagnosis of Bacterial diseases.
(Sample collection, transportation, bacterial identification methods in
2.1 general)
2
Demonstration of respect for patient samples sent for laboratory
2.2 investigations
Assessment
2.3 Hand hygiene, PPE- Donning & Doffing of hand gloves (1)
3
Skill Certification
3.1 Direct methods of bacterial detection - Simple staining & Hanging drop
Assessment 4
3.2 Gram staining (1)
Skill Certification
4 ZN Staining (1)
5
Skill Certification
Bacterial culture: Culture media, methods and identification techniques
5 (conventional and automated) 6
Assessment
6 Antibiotic sensitivity testing
7
Assessment
Indirect methods of infectious disease diagnosis
7 (Immunological diagnostic tests) 8
Assessment
8 General principles of laboratory diagnosis of Viral diseases
9
Assessment
9.1 General principles of laboratory diagnosis of Parasitic diseases
10
Assessment
9.2 Stool Examination (1)
11
Skill Certification
10 General principles of laboratory diagnosis of Fungal diseases
12
Assessment
221
Topics, Exercises and Assessment
S.No 2.00 Hours
Hospital infection control – I: Hand hygiene, PPE (Donning &
11 Doffing) (2) 13
Skill Certification
Hospital infection control - II: Sterilization & Disinfection, Biomedical
12 waste management, Needle stick injuries 14
Assessment
13.1 Laboratory diagnosis of Infective endocarditis
13.2 Laboratory diagnosis of Rheumatic heart disease
Assessment 15
13.3 Laboratory diagnosis of Sepsis + Gram Staining (2)
Skill Certification
14.1 Laboratory diagnosis of Brucellosis
14.2 Laboratory diagnosis of Leptospirosis 16
Assessment
14.3 Laboratory diagnosis of Scrub typhus
14.4 Laboratory diagnosis of Dengue fever
17
14.5 Laboratory diagnosis of Candidemia
Assessment
15 Laboratory diagnosis of Enteric fever
18
Assessment
16.1 Laboratory diagnosis of Falciparum Malaria
16.2 Laboratory diagnosis of Vivax Malaria 19
Assessment
17.1 Laboratory diagnosis of visceral Leishmaniasis
17.2 Laboratory diagnosis of lymphatic Filariasis 20
Assessment
18.1 Laboratory diagnosis of HIV infection
18.2 Confidentiality pertaining to patient's identity in laboratory result 21
Assessment
19.1 Laboratory diagnosis of cholera
19.2 Laboratory diagnosis of Rotaviral Diarrhea
19.3 Laboratory diagnosis of antibiotic associated diarrhea 22
19.4 Laboratory diagnosis of Giardiasis
Assessment
222
S.No 2.00 Hours
19.5 Laboratory diagnosis of Cystisosporiasis/Cryptosporidiosis
19.6 ZN Staining (2)
Skill Certification 23
19.7 Laboratory diagnosis of helminthic diarrhea + Stool examination (2)
Skill Certification
20.1 Laboratory diagnosis of Bacillary dysentery
Assessment
24
20.2 Laboratory diagnosis of Amoebic dysentery + Stool examination (3)
Skill Certification
21.1 Laboratory diagnosis of Ascariasis
21.2 Laboratory diagnosis of Hymenolepiasis
Assessment
25
Laboratory diagnosis of asymptomatic intestinal heminthic infections +
21.3 Stool examination (4)
Skill Certification
21.4 Laboratory diagnosis of Strongyloidiasis
21.5 Laboratory diagnosis of Enterobiasis
Assessment 26
21.6 Laboratory diagnosis of Hookworm infection + Stool examination (5)
Skill Certification
III Infections of hepatobiliary system
22.1 Laboratory diagnosis of Hepatitis A
22.2 Laboratory diagnosis of Acute Hepatitis B
22.3 Laboratory diagnosis of Chronic Hepatitis B
27
22.4 Laboratory diagnosis of Hepatitis C Carrier
22.5 Laboratory diagnosis of Hepatitis E
Assessment
22.6 Laboratory diagnosis of Amoebic liver abscess
22.7 Laboratory diagnosis of Hydatid cyst of liver 28
Assessment
23.1 Laboratory diagnosis of Breast abscess + Gram staining (3)
Skill Certification
23.2 Laboratory diagnosis of Furuncle
29
23.3 Laboratory diagnosis of Gas gangrene
23.4 Laboratory diagnosis of cellulitis
Assessment
223
S.No 2.00 Hours
23.5 Laboratory diagnosis of wound infection
23.6 Laboratory diagnosis of burns wound infection 30
Assessment
23.7 Laboratory diagnosis of Leprosy
23.8 ZN Staining (3)
Skill Certification
24.1 Laboratory diagnosis of Herpes labialis 31
24.2 Laboratory diagnosis of Measles
24.3 Laboratory diagnosis of Molluscumcontagiosum
Assessment
24.4 Laboratory diagnosis of Tineacororis
24.5 Laboratory diagnosis of Mycetoma
24.6 Laboratory diagnosis of Mucormycosis 32
24.7 Laboratory diagnosis of Oral thrush
Assessment
25.1 Laboratory diagnosis of Septic arthritis
25.2 Laboratory diagnosis of Gonococcal arthritis
25.3 Laboratory diagnosis of Acute osteomyelitis
33
Assessment
25.4 Laboratory diagnosis of Tubercular osteomyelitis + ZN staining (4)
Skill Certification
26.1 Laboratory diagnosis of Aseptic meningitis
26.2 Laboratory diagnosis of Cryptococcal meningitis
26.3 Laboratory diagnosis of Pyogenic meningitis + Gram staining (4)
34
Skill Certification
27.1 Laboratory diagnosis of encephalitis
Assessment (26.1, 26.2, 27.1)
28.1 Laboratory diagnosis of Diphtheria + Gram staining (5)
Skill Certification
29.1 Laboratory diagnosis of Pneumococcal pneumonia
Assessment
35
29.2 Laboratory diagnosis of Pulmonary Aspergillosis
Assessment
29.3 Laboratory diagnosis of Pulmonary tuberculosis + ZN staining (5)
Skill Certification
29.4 Hand hygiene, PPE (3)
36
Skill Certification
S.No Topics, Exercises and Assessment 2.00 Hours
224
30.1 Laboratory diagnosis of Primary Syphilis
30.2 Laboratory diagnosis of Herpes genitalis
37
30.3 Laboratory diagnosis of Trichomoniasis
Assessment
31.1 Urinary tract infection
38
Assessment
225
BLOCK I: General Microbiology,
Immunology & Hospital Infection Control
1 A. Introduction to Microbiology Laboratory
Suggested Teaching-Learning methods

Small Group Discussion
 Department tour to visit various sections of the laboratory
 Demonstration of Koch’s postulates (using models, charts, cultures etc.), Universal safety
precautions, and good laboratory practices (using live demonstrations, videos and charts etc.)
 Demonstration of different types of Biosafety cabinets with suitable examples of handling
bacteria based on their biohazard class.
 Demonstration and practice of Hand hygiene and, donning & doffing of gloves
1-B. Microscopy
 Small Group Discussion: Using suitable Case Scenario based/Problem based exercise ( E.g:
Stool examination for parasitic eggs, Bacterial examination of body fluids)
 DOAP/ Demonstration of:
- Handling and focusing slides/preparations under various magnifications using suitable
case scenarios and clinical samples
- Interpretation of findings
- Relative measurement of various microscopic objects
226
2- General Principles of Laboratory Diagnosis of Bacterial diseases.
(Sample collection, transportation, bacterial identification methods in general, **Hand hygiene,

PPE- Donning & doffing of hand gloves-1, *Demonstrate respect for patient samples sent for
laboratory investigations)

 Small Group Discussion: Using suitable Case Scenario based/Problem based exercise (Eg:
A patient admitted with 70% burns develops wound infection and high fever on the fourth
day, discuss the clinical diagnosis, probable causative microorganisms and suggest suitable
laboratory tests in this case)
 Demonstration on,
- Selection & collection of suitable clinical sample/s aseptically based on the case scenario
(Pus and blood samples for culture in this case) using the models/manikins
- How to fill up the sample request form with necessary data
- General steps involved in the laboratory diagnosis of bacterial diseases
 DOAP: on Hand hygiene and donning and doffing of hand gloves
 SGD, Role Play, Role modeling, video etc. for AETCOM session on how to respect patient
samples sent to the laboratory for detection of microbial agents causing Infectious diseases.
(e.g: A case scenario requiring ethical and moral obligations. Here one can demonstrate, what
may happen when there is breech in professional conduct pertaining to the clinical samples)
227
(Source: Center for Disease Control, Atlanta, USA)
Exercise 2.2:Demonstrate respect for patient samples sent to the laboratory for
performance of laboratory tests in the detection of microbial agents causing infectious
diseases.
Suggested Teaching-learning method:
 Aninteractive session on ways to demonstrate respect for clinical sample

 Develop case scenarios/ problem solving exercises for small group discussions designed to
highlight ways that show disrespect to sample E.g. situations highlighting inappropriate
container/ site/ sample/ amount, label, incomplete or wrong information in request form, time
of transportation, storage condition, turnaround time, quality of test performance, test result
etc. and ways to correct them. Also highlight the impact these events will have on clinical
outcome of patients.
 Role play by the students.
 Wrap up session
 Reflective writing and summary of key learning in log book
228
3 - Direct methods of bacterial detection
(Simple staining, Hanging drop, *Gram staining)
 Small Group Discussion: Brief discussion on a) types of staining b) principles of simple
staining, hanging drop and Gram staining, using appropriate case scenarios, pictures, charts,
focused microscopic slides/preparations, videos etc.
(e.g: Case scenario of urethritis for demonstration of Neisseria by simple staining by
methylene blue , A case of diarrhea with rice water stools with severe dehydration for
handing drop to demonstrate darting motility of V. cholerae, and a case of wound infection
for Gram stain to demonstrate Gram positive cocci)
 Demonstration/DOAP on: Making smear from clinical/simulated sample, heat fixing the
smear and staining the slide by Gram staining
 Performance of :Gram staining, focusing the stained slides & interpretation of results by
students followed by Certification.
4 – Ziehl-Neelsen staining
 Small Group Discussion: Brief discussion on a) Acid fastness and acid fast organisms, b)
principle of Ziehl-Neelsen’s stain c) Application of Ziehl-Neelsen’s stain in appropriate
clinical conditions
(By using suitable case scenarios, pictures, charts, focused microscopic slides/preparations,
videos etc.)
( Eg: An adult male presenting with evening rise of fever, productive cough with
hemoptysis, loss of appetite for three weeks, can be used to discuss the differential
diagnosis, arrive at clinical diagnosis, select the right laboratory test, instruct the patient for
proper collection of clinical specimen and perform Ziehl-Neelsen’s staining.)
 Demonstration/DOAP on: Making smear from clinical/simulated sample, heat fixing the
smear and staining the slide by Ziehl-Neelsen’s staining
 Performance of:Ziehl-Neelsen’s stain, focusing the stained slides & interpretation of
results by students followed by Certification.
229
5-Bacterial culture
(Media, methods and identification -conventional and automated)
 Small Group Discussion: Brief discussion on a) Rationale of growing the bacteria in the
laboratory b) Classification and types of culture media c) selection of culture media based
on the type of disease and clinical specimen
(By using suitable case scenarios, Eg:Surgical site infection, Septicemia, Urinary tract
infection, diarrhea, dysentery, respiratory tract infections for demonstration of different
culture media like, Blood agar, Chocolate agar, Mac.Conkey’s agar, Blood culture bottles
(conventional and automated), CLED medium, Selenite F broth, DCA, XLD, TCBS,
Loffler’s serum slope, RCM etc. appropriately)
 Demonstration on :
- Inoculation of clinical specimen on suitable culture media
- Aerobic and anaerobic culture methods
- Presumptive identification tests (Cholera red reaction etc.)
- Important colony characteristics (alpha/beta hemolytic colonies, lactose fermenting/non
lactose fermenting colonies on Mac.Conkey agar etc.)
- Key biochemical reactions used in presumptive identification of bacteria (Coagulase,
catalase, Oxidase &IMViC tests)
230
6- Antibiotic sensitivity testing
Suggested Teaching-Learning methods:
Small Group Discussion: Brief discussion on,

 Emerging menace of multidrug resistance by using a clinical case scenario,
(A case of chronic non-healing ulcer, chronic urinary tract infection etc.)
 Methods available for antimicrobial susceptibility testing, selection of method with
briefing on their advantages and limitations
 Interpretation of the results
Demonstration of,
 Antimicrobial susceptibility tests (Kirby Bauer Disc diffusion, MIC, Epsilometer/E-Test)
 Interpretation of the results of antimicrobial susceptibility testing
 Recommendations for antimicrobial therapy
7- Indirect methods of disease diagnosis (Immunological tests)

Small Group Discussion: Brief discussion on,
a) Basis of immunological tests, types of antigen-antibody reactions and their application in
diagnosis of the infectious diseases.
b) Types of agglutination tests and choosing the right test in a given clinical scenario
(Eg 1: A child with suspected rheumatic fever to demonstrate how to screen for Anti-streptolysin
O by latex agglutination test.)
(Eg 2: A case of Pyrexia of unknown origin in the second week of illness to explain how to
utilize Widal test to diagnose typhoid fever)
c) Types of Precipitation tests and choosing the right test in a given clinical scenario
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(Eg: Person with painless ulcer on the genital area with history of sexual exposure, to learn how
to screen the cases for syphilis using VDRL test)
d) Principle of ELISA and its utilization mainly for the diagnosis of viral infections
(Eg 1: A patient on long term renal dialysis with jaundice suspected of Hepatitis B infection for
demonstration of antigen detection ELISA.)
(Eg 2: A truck driver with history of sexual exposure, chronic diarrhea, weight loss, suspected of
HIV infection to demonstrate antibody detection ELISA)
e) Principle of Immunofluorescence test and its applications
(Eg 1: Antibody detection in a case of tuberculosis)
(Eg. 2: A person with collagen vascular disorder, for screening of Antinuclear antibodies)
11) Rapid tests (flow through, lateral flow, solid phase assays using cards, cassettes, strips etc.)
and their application with advantages and limitations of such tests
(Eg 1: A victim of severe road traffic accident requiring emergency screening for
infectious diseases before surgery and blood transfusion)
8- General principles of Laboratory diagnosis of Viral diseases

Discussion: A case based discussion on different causative agents of viral infections/ diseases,
the need and the methods used for laboratory diagnosis of common viral infections.
( A case of herpetic lesions on the face, A child with rotaviral diarrhea etc)
Demonstration: Sample collection for viral diagnosis, Viral transport medium (VTM), electron
microscopic pictures and models of viruses, specimen of chorio-allantoic membrane with
lesions, Immunofluorescence staining, histopathological staining showing inclusion bodies
(Slide/Pictures), PCR set up, ELISA (for antigen and antibody detection), immuno-
chromatographic and rapid tests.
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9 -General principles of Laboratory diagnosis of Parasitic diseases

Discussion : A case based discussion on different parasites causing human infections.
Need for laboratory diagnosis of parasitic infections
Methods used in the laboratory diagnosis.
( Eg: A case of amoebic dysentery, A case of fever, chills (malaria) etc.)
Demonstration: Sample collection and transportation for diagnosis of parasitic infections, stool
collection device, gross examination of specimen, preparation of saline and iodine mount from
stool sample, various ova and cysts of common parasites in the stool, plasmodia and other
protozoal forms from peripheral smear (photographs can be used in case of uncommon
parasites), ELISA, immuno-chromatographic and rapid tests.
10 - General principles of Laboratory diagnosis of Fungal diseases

Discussion : A case based/problem based discussion on different causative agents of fungal
infections/ diseases, the need and the methods used for laboratory diagnosis of common fungal
infections.
( Eg: A case ringworm infection, mucocutaneous candidiasis, etc)
Demonstration: Sample collection for fungal diagnosis, Microscopic slide mount of KOH
preparation, Gram staining of Candida and Cryptococcus species, negative staining for
Cryptococcus species, lactophenol cotton blue mount (LPCB) of Aspergillus, Sabouraud
dextrose agar (SDA) with and without growth of common yeast and molds, slide culture etc.
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11 - Hospital infection control I: Hand hygiene, PPE (Donning & doffing)

DOAP session and Small Group Discussion
• Demonstration of steps of hand rub, hand wash and hand scrub
• Demonstration of donning of various personal protective equipment (PPE) used in in
• Demonstration of doffing of various personal protective equipment (PPE) used in in
12 - Hospital infection control II: Sterilization & Disinfection,

Biomedicalwaste management, Needle stick injuries

Small Group Discussion and Demonstration
• Demonstration of the common sterilants and disinfectants used in healthcare settings and
their clinical applications
• Department tour to central sterile supply department (CSSD)
• Demonstration of segregation of common biomedical waste generated in a healthcare
setting
• Demonstration of the steps of blood spill management
• Demonstration of respiratory hygiene and cough etiquette
• Demonstration of the methods used and significance of assessing the microbial
contamination of food, water and air
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BLOCK II: Bloodstream and CVS Infections
13 - Laboratory diagnosis of Rheumatic heart disease, Infective endocarditis

and Sepsis

• Small group discussion based on clinical case scenario
• Demonstration of methods of laboratory diagnosis for identification etiologic agents of
infective endocarditis (e.g, Viridans Streptococci, Staphylococcus aureus, Coagulase
negative Staphylococcus Spp)
• Demonstration of methods of laboratory diagnosis for acute rheumatic fever (e.g, ASLO)
Exercise 13.1: Infective endocarditis

Clinical case: A case of fever, weakness and valvular lesions
Key words for case scenario: Fever, severe back-pain and weakness in lower limbs, non-tender,
small erythematous nodular lesions on soles, vegetations on mitral valve, blood cultures
Exercise 13.2: Acute Rheumatic fever

Clinical case: A case of migrating joint pains, subcutaneous nodules and murmur
Key word: Swollen, red, and/or tender joints, which migrates from one joint to another over a
period of hours, abnormal gait, painless mobile lumps beneath the skin of hands, feet, and
elbows, murmur heard over the mitral valve area, prolongation of P-R interval, episode of sore
throat 3 weeks back.
Exercise 13.3: Sepsis

Clinical case: A case with fever, low BP and positive blood culture
Key words for case scenario: Body temperature increased, heart rate increased and respiratory
rate increased, blood pressure decreased and urine output was significantly decreased, blood
culture flagged positive
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14. Laboratory diagnosis of Brucellosis, Leptospirosis, Dengue fever &
Chikungunya, scrub typhus and Systemic candidiasis

DOAP Session and Small group discussion
• Demonstration of laboratory methods for the etiological diagnosis of brucellosis
• Demonstration of laboratory methods for the etiological diagnosis of leptospirosis
• Demonstration of laboratory methods for the etiological diagnosis of scrub typhus
• Demonstration of laboratory methods for the etiological diagnosis of dengue
haemorrhagic fever
Demonstration of laboratory methods for the etiological diagnosis of systemic candidiasis
Exercise 14.1: Brucellosis

Clinical case: A case of intermittent fever, organomegaly, positive blood culture
Key words for case scenario: On and off fever with profuse night sweats and joint pain,
hepatosplenomegaly, blood culture, standard agglutination test, animal exposure (e.g. raw milk
intake
Exercise 14.2: Leptospirosis

Clinical case: A rice-field worker with jaundice, organomegaly, oliguria and fever
Key words for case scenario: Fever, yellow discoloration of skin and sclera,
hepatosplenomegaly, neutrophilia, thrombocytopenia, elevated conjugated bilirubin with mild
elevation of transaminases, oliguric and uremic, farmer, rice field, rainy season
Exercise 14.3: Scrub typhus

Clinical case: A case of skin rashes with eschar
Key words for case scenario: Maculopapular rashes, lymphadenopathy and eschar on upper
limb, fever, myalgia and respiratory distress, Weil Felix test
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Exercise 14.4: Dengue
Clinical case: A case of fever, joint pain, rash and thrombocytopenia
Key words for case scenario: Fever, severe joint pain, back pain and myalgia, petechial rashes
over the body, jaundice, hepatomegaly and a low platelet count, positive tourniquet test, history
of mosquitoes bite, NS1 antigen
Exercise 14.5: Systemic candidiasis

Clinical case: An HIV patient with fever and hypotension, budding yeast cells in blood culture
Key words for case scenario: Immunosuppression (e.g. HIV-infected or febrile neutropenia),
fever and altered mental status, low blood pressure, increased respiratory rate, blood cultures,
budding yeast cells
15 - Laboratory diagnosis of Enteric fever

Demonstration of methods of laboratory diagnosis for identification of etiologic agents of enteric
fever (Key culture tests, biochemical tests, antibiogram and serological tests)
Exercise 15.1: Enteric fever

Clinical case: A case of fever and splenomegaly with positive blood culture
Key words for case scenario: High-grade fever, abdominal pain, nausea, vomiting and
anorexia, coated tongue, palpable spleen, automated culture bottle, Widal test
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16 - Laboratory diagnosis of Malaria
• Demonstration of methods of laboratory diagnosis for their identification of etiologic
agents of malaria
Exercise 16.1: Falciparum malaria
Clinical case: A case of fever with chills and rigors, splenomegaly
Key words for case scenario: Odisha, fever, chills and rigor, anemia, seizures, splenomegaly,
peripheral blood smear examination
Exercise 16.2: Vivax malaria

Clinical case: A case of fever with chills and rigors with splenomegaly and anemia
Key words for case scenario : Uttar Pradesh, fever, chills and rigor, anemia, splenomegaly,
17 - Laboratory diagnosis of Filariasis and other blood parasites (Leishmania,

Trypanosomes)
 Small group discussion based on clinical case scenario and demonstration
 Demonstration of methods of laboratory diagnosis of kalaazar
 Demonstration of methods of laboratory diagnosis for lymphatic filariasis
Exercise 17.1: Visceral leishmaniasis

Clinical case: A case with fever, organomegaly and hyperpigmentation
Key words for case scenario: Bihar, splenomegaly, anemia and fever, hyperpigmentation, bone
marrow aspirate, LD bodies
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Exercise 17.2: Lymphatic filariasis
Clinical case: A case of fever and unilateral limb swelling
Key words for case scenario: Fever on and off, unilateral swelling of the left lower limb,
18 - Laboratory diagnosis of HIV infection

(*Confidentiality pertaining to patient's identity in lab result)
Small group discussion based on clinical case scenario and demonstration
• Demonstration of methods of laboratory diagnosis of HIV
• Video demonstration/Role play/OSCE for demonstration of maintenance of
confidentiality pertaining to laboratory test results
Exercise 18.1: AIDS
Clinical case: A promiscuous man with chronic diarrhea and lymphadenopathy
Key words for case scenario: Male with history of multiple sex partners, unexplained fever,
progressive loss of weight, persistent diarrhea and generalized lymphadenopathy
Exercise 18.2: Demonstrate confidentiality pertaining to patient identity on laboratory
results.
 Have a brainstorming session with students to introduce the patients’ rights along with
ethical, sociocultural and legal aspects of confidentiality in laboratory.
 Develop case scenario and conduct small group discussions/ role play with studentsto help
them identify security threats and vulnerability of laboratory data along withthe impact of
such events on patients’ trust/outcome and suggest ways to rectify them.
 Have a wrap up session
 Ask students to write reflections and summary of learning in logbook
Exercise /activity:
 Design situations/case scenario that help students to identify the events that breach
confidentiality of dataeg. situations highlighting unauthorized access, use, disclosure,
modification, loss or theft of laboratory data
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BLOCK III: Gastrointestinal and
Hepatobiliary Infections
19 - Laboratory diagnosis of diarrhea

Small group discussion based on clinical case scenario and demonstration to assess
 Interpretation of the case and coming up with differential diagnosis
 Choosing appropriate sample and describing sample collection and transportation procedure
 Identifying the causative pathogen based on the history, microscopy, cultural and
biochemical characters/ photographs, laboratory investigation reports, etc
 Suggesting tests to confirm the diagnosis and also to refute other organisms chosen in
differential diagnosis
 Choosing appropriate treatment and suggesting specific prophylactic measures
 Interpreting stool examination and culture reports in a case of diarrhea
A. Stool examination for parasites – DOAP
B. Hanging drop preparation – Demonstration
Exercise 19.1: Cholera

Clinical case: An outbreak of acute diarrhea with dehydration
Key words for Case scenario: Adult, outbreak of diarrhea, rice water stools, signs of severe
dehydration, investigations with stool sample, Stool culture – key culture media and
biochemical, Antibiogram
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Exercise 19.2: Rota virus diarrhea
Clinical case: A child with diarrhea
Key words for Case scenario: Child, diarrhea, fever, signs of dehydration, No parasites
demonstrated in stool wet mount, No bacterial pathogen isolated from stool culture, stool
microscopy inconclusive, significant finding in electron microscopy of stool is provided
Exercise 19.3: Antibiotic associated diarrhea
Clinical case: Diarrhea in a person taking antibiotics

Key words for Case scenario: Adult with upper respiratory infection, broad spectrum antibiotic
for extended period, stool routine, Toxin and Glutamate dehydrogenase antigen detected in stool
sample by rapid test
Exercise 19.4: Giardiasis
Clinical case: A case of chronic diarrhea, foul smelling stools with mucus
Key words for Case scenario: Adult, increased frequency and liquid stools, foul smelling stool,
stool examination, stool wet mount with Giardia cyst is focused/ picture
Exercise 19.5: Cystisosporiasis/Cryptosporidiasis
Clinical case: An AIDS patient with persistent diarrhea

Key words for Case scenario: AIDS patient with chronic watery diarrhea, Features of severe
malnutrition, stool examination, modified acid fast stained smear/picture of parasite provided
Exercise 19.7: Diarrhea due to helminthic infection- Ascariasis/,

Trichuriasis/Hymenolepiasis, etc.
Clinical Case: A case of diarrhea and abdominal discomfort
Key words for case scenario: Adult, frequent loose stools of short duration, abdominal
discomfort, no blood, stool examination
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20 - Laboratory diagnosis of dysentery
- Interpretation of the case and coming up with differential diagnosis
- Choosing appropriate sample and describing sample collection and transportation
procedure
- Identifying the causative pathogen based on the history, microscopy, cultural and
- Suggesting tests to confirm the diagnosis and also to refute other organisms chosen in
- Choosing appropriate treatment and suggesting specific prophylactic measures
- Interpreting stool examination and culture reports in a case of dysentery
 Stool wet mount examination – DOAP
Exercise 20.1: Bacillary dysentery

Clinical case: A Case of frequent blood-stained stools
Key words for case scenario: Increased frequency (>10 times per day) of passing small quantity
stools, fresh blood seen in the stool, tenesmus,
Stool culture findings and key biochemical reactions
Exercise 20.2: Amoebic dysentery

Clinical case: A case of frequent dark red offensive-smelling stools
Key words for case scenario: Adult, increased frequency (6-8 times per day) of passing copious
offensive smelling stools, dark red colour, tenesmus, fever.
 Demonstration of Entamoeba histolytica cysts or pictures of cysts/trophozoites
 Emphasis on differentiating amoebic and bacillary dysentery
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21 - Laboratory diagnosis of intestinal helminthic infections

procedure
- Identifying the causative pathogen based on the history, macroscopy, microscopy,
photographs, laboratory investigation reports, etc
- Choosing appropriate treatment
 Demonstration of adult parasites, Ova
 Stool examination – DOAP
Exercise 21.1: Ascariasis/ Intestinal Taeniasis

Clinical case: A child who passes a motile fleshy thing in stools
Key words for case scenario: Child, passed in stools - white motile worm with cylindrical body
OR white flat fleshy motile structure
Demonstration:Specimen of round worm or tapeworm adult
Exercise 21.2: Hymenolepiasis

Clinical Case: A case of abdominal pain and malnourishment
Key words for case scenario: Abdominal pain, diarrhea, malnourishment, stool examination
Exercise 21.3: Asymptomatic intestinal helminthic infection – Ascariasis/Trichuriasis

Clinical case: An incidental stool finding in apparently normal individual
Key words for case scenario: An asymptomatic adult, routine stool examination as part of
annual health check-up
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Performance (DOAP):Stool examination exercise
Exercise 21.4: Strongyloidiasis

Clinical case: A diabetic with persistent loose stools
Key words for case scenario: Adult, uncontrolled diabetes, pain abdomen, diarrhea, fever, Stool
examination (Demo of stool wet mount with larva/picture)
Exercise 21.5: Enterobiasis

Clinical case: A child with perianal itching
Key words for Case scenario: Child, perianal itching worse at night, small white worms noted
in the inner garments of the child, stool routine inconclusive
Demonstration:NIH swab, picture of ova, discussion on sample collection method,
demonstration of adult worm specimen/picture
Exercise 21.6: Hook worm infection

Clinical case: A case of anemia
Key words for Case scenario: Farmer, from village with practice of open air defecation,
tiredness and breathlessness on exertion, pallor
Performance (DOAP): Stool examination exercise
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22. Laboratory diagnosis of hepatic infections

 Small group discussion based on clinical case scenario
- Analyzing the case scenario and arriving at clinical/differential diagnosis
- Suggesting appropriate sample and its collection method
- Interpretation of charts/diagnostic test reports
- Demonstration of laboratory tests like immunochromatography, ELISA, etc
- Suggesting specific prophylaxis when applicable
Exercise 22.1: HAV hepatitis

Clinical case: An outbreak of fever and jaundice
Key words for Case scenario: Child, fever, malaise, loss of appetite, icterus, tender
hepatomegaly, high coloured urine, two more people in the locality have similar complaints in
the same week, Blood investigations, HAV IgM positive
Exercise 22.2: Acute HBV hepatitis

Clinical case: An IV drug abuser with jaundice
Case scenario: Young adult, IV drug use, sharing of needles, acute fever, malaise, jaundice, loss
of appetite, pain abdomen, hepatomegaly, HBsAg positive
Exercise 22.3: Chronic viral hepatitis B

Clinical case: A case of fever with jaundice for a few months
Key words for Case scenario: Adult, fever and icterus since a few months, loss appetite, tender
hepatomegaly, elevated liver enzymes, HBsAg (+), Anti-HBcIgG (+), HBeAg (-)
Exercise: 22.4: Hepatitis C carrier

Clinical Case: An asymptomatic person with an abnormal liver seromarker
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Key words for Case scenario: Adult, asymptomatic, routine annual health check-up, anti-HCV
IgG positive by ELISA
Exercise 22.5: Acute Hepatitis-E

Clinical case: A pregnant woman with jaundice
Key words for Case scenario: Pregnant woman in 2nd trimester, fever and jaundice, loss of
appetite, malaise, raised liver enzymes, HEV IgG positive by ELISA
Exercise 22.6: Amoebic liver abscess

Clinical case: A case of tender hepatomegaly
Key words for Case scenario: Adult, acute onset fever, pain abdomen, tender hepatomegaly,
past history of dysentery, USG showed hypoechoic lesion in right lobe, reddish brown purulent
material drained by USG guided aspiration, microscopy showed the structures, stool examination
showed no abnormal findings
Exercise 22.7:Hydatid cyst of the liver

Clinical case: A case of cystic hepatic lesion
Key words for Case scenario: Adult, shepherd, pain in right upper abdomen, no jaundice, USG
shows single cystic lesion, removed by surgery
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BLOCK IV: Skin, Soft Tissue and
Musculoskeletal System Infections
23. Laboratory diagnosis of bacterial skin infections

procedure
- Identifying the causative pathogen based on the history, microscopy, cultural and
- Choosing appropriate treatment and suggesting specific prophylactic measures
- Interpreting antibiotic susceptibility data and deciding the most appropriate antibiotic in a
given situation (e.g, MRSA)
 Sill Assessment: Gram stain and Acid fast staining – DOAP
Exercise 23.1: Breast abscess

Clinical case: A lactating mother with painful breast swelling
Key words for Case scenario: Young Adult lady, painful swelling on left breast, fever, redness
and tenderness, suitable sample collected and following tests performed
DOAP:Gram stain exercise – Staphylococcus aureus
Exercise 23.2: Furuncle

Clinical case: An young man with boils on the leg
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Key words for Case scenario: Young adult, painful lesions on the leg with pus pointing,
appropriate sample was collected and the following tests were performed
Exercise 23.3: Gas gangrene
Clinical case: A person with discoloured limb with bubbles following crush injury
Key words for Case scenario: Road traffic accident, Crush injury to limb, pain, foul-smelling
serosanguinous discharge, crepitus/gas bubbles, shock, Microscopy shows box-car-shaped gram
positive bacilli
Demonstration of pictures of clinical features and microscopy of the pathogen
Exercise 23.4: Cellulitis by Streptococcus pyogenes

Clinical case: A diabetic with painful swelling of leg
Key words for Case scenario: Diabetic, swollen painful leg, reddish skin, ill-defined border,
minimal thin watery discharge, fever, pain, culture and sensitivity performed
Exercise 23.5: Wound infection

Clinical case: Wound infection (Staphylococcus aureus, E.coli, Klebsiella, Enterococcus,
Proteus Spp, Acinetobacter Spp., Pseudomonas aeruginosa)
Key words for Case scenario: Adult, large wound on right thigh following road traffic accident,
developed purulent discharge and fever, culture and sensitivity performed
Exercise 23.6: Infection of burns wound

Clinical case: Burns wound with discharge
Key words for Case scenario: Adult, 10% burns involving chest and left arm, fever and
greenish purulent discharge from the burns ulcers, culture and sensitivity performed
Exercise 23.7: Leprosy

Clinical case: A case of hypoesthetic skin patch
Key points for case scenario: Adult, hypopigmented skin patches, face and chest, decreased
sensation, no itching, Smear prepared from the lesion is focused under microscope
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Exercise 23.8:ZiehlNeelsen stain exercise with abdominal tuberculosis case scenario
24 - Laboratory diagnosis of fungal and viral skin infections

- Analysing case scenario/clinical photograph and arriving at clinical/differential diagnosis
- Choosing appropriate sample and test to be performed
- Interpreting laboratory test results
- Identifying pathogen based on typical culture characters and microscopic findings –
actual/pictures (Eg,.Tzanck smear for Herpes; Fungi – growth on SDA and Gram
stain/LPCB mount)
 Acid fast staining – DOAP
Exercise 24.1: Localized Vesicular skin lesions – herpes labialis

Clinical case: A case of painful perioral lesions with fever
Key points for Case scenario: Adult, painful grouped vesicular lesions on lip, fever, similar
lesions in the past associated with fever, smear prepared from the sample from the lesions
Exercise 24.2: Measles

Clinical case: A case of generalized exanthematous skin lesions and fever
Key points for Case scenario: Unimmunized child, dry cough, fever and maculopapular rash,
no vesicles, developing from head to trunk then on limbs, no lesions of palms and soles, white
lesions on buccal mucosa opposite to upper 1 st molar, Picture of the skin rash and its distribution
provided
Exercise 24.3: Molluscumcontagiosum

Clinical case: A case of multiple umbilicatedpapular lesions
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Key words for Case scenario: Child, multiple waxy papular lesions with umbilication, painless,
face (Picture)
Exercise 24.4: Tineacorporis

Clinical case: A case of itchy ring-shaped skin lesions
Key words for Case scenario: Adult, itchy, ring-shaped skin lesions on chest and abdomen,
scaly, central clearing, Microscopy and Culture tests done and provided
Exercise 24.5: Mycetoma foot

Clinical case: A case of swollen foot with multiple discharging openings
Key words for Case scenario: Adult, trauma, tumour-like swelling on foot, multiple
discharging sinuses, granules, minimal pain, long duration, Histopathology of biopsied
tissue/crush smear from the granule
Exercise: 24.6: Rhino-orbital mucormycosis

Clinical case: A diabetic with blackish skin lesions on face
Key points for Case scenario: Elderly, Diabetic, on irregular treatment, blackish discolouration
of facial skin around eye and nose, fever, fruity odour in breath, high blood sugar level, ketone
bodies in urine.
Demonstration:Growth on SDA and LPCB mount provided
Exercise 24.7: Oral thrush

Clinical case: A cancer patient with white oral lesions
Key points for Case scenario: Adult with malignancy, on chemotherapy, white patches on oral
mucosa, tests performed to diagnose the condition displayed (Gram stain and growth on SDA)
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25. Laboratory diagnosis of musculoskeletal infections
Exercise25.1: Septic arthritis – S.aureus
Clinical case: A case of painful swollen joint with fever
Case scenario: Adult, sudden onset pain in the right knee, fever, knee is swollen and tender,
restricted movements, pus aspirated from the knee and culture performed, tests displayed, culture
and sensitivity done
Exercise 25.2: Septic arthritis – Gonococcus

Clinical case: A case of painful swollen joint and skin rashes
Key points for Case scenario: Young man, history of purulent discharge per urethra and
burning micturition in the recent past, fever and malaise, left knee swollen and tender,
movements restricted, erythematous skin lesions on trunk, Pus aspirated and tests performed and
displayed, Microscopy done
Exercise 25.3: Acute osteomyelitis

Clinical case: A case of painful swollen limb with fever after trauma
Key points for Case scenario: Child, history of trauma, open wound just above the ankle,
developed pain and swelling, fever, purulent discharge, culture and sensitivity
Exercise 25.4: Chronic vertebral osteomyelitis – TB

Clinical case: An adult with chronic backache and weight loss
Key points for Case scenario: Adult, history of low backache, weight loss, bone tenderness at
Lumbar vertebrae, Abnormal X-ray findings (lytic), Aspirated material investigated, Culture
done
DOAP:Ziehl-Neelsen stain – certifiable exercise
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BLOCK V: Central nervous system infections
26. Laboratory diagnosis of Meningitis
Suggested Teaching-Learning Methods
 Small Group Discussion: Using suitable Case Scenario or laboratory reports of patients
diagnosed with specific CNS infection ( Eg Pyogenic meningitis in a child, an AIDS
patient with chronic headache)
 Demonstration of microscopic slides of CNS pathogens like
 Meningococci, Pneumococci, H.Influenzae, Toxoplasma gondii, Trypanosoma
 India ink preparation of Cryptococcus neoformans
Exercise 26.1:Aseptic Meningitis

Clinical Case: A febrile drowsy and irritable child
Key points for case scenario: boy, two days history of fever, lethargy, irritability and poor
feeding, febrile and drowsy, no neck stiffness
Exercise 26.2:Cryptococcal meningitis

Clinical Case: An AIDS patient with fever, headache and stiff neck
Key points for case scenario: Adult, headache, fever, vomiting and stiff neck, CSF: low
glucose, high protein and PMNs <300/mm3, Indian ink- capsulated, budding yeast cells
Exercise 26.3:Pyogenic meningitis

Clinical Case: A case of fever, headache and stiff neck
Key points for case scenario: Child, high grade fever, vomiting, altered consciousness and neck
stiffness, CSF- plenty of pus cells and short gram negative pleomorphic bacilli
Exercise27.1: Encephalitis
Clinical case: A case of fever and seizure
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Key points for Case scenario: Child, seizures, drowsy, fever, headache and irritable, neck
stiffness absent, rashes, probable clinical diagnosis of encephalitis
BLOCK VI: Respiratory tract infections

28. Laboratory diagnosis of Upper respiratory tract infections (URTI)
 Case based exercises with prepared smear for reporting by individual students
 Role play for instructions to be given to patient for sputum collection
 Small group case discussion for key difference in clinical presentation, etiological agent,
choice of sample collection, microbiological investigation for Upper Respiratory tract
infection and interpretation of Laboratory reports
 Demonstrations of upper respiratory pathogens like C. diphtheriae (in Gram stain, Albert
stain), Staphylococcus aureus, Streptococcus pyogenes
Exercise 28.1: Diphtheria

Clinical Case: A febrile child with white membrane in throat
Key points for case scenario: unimmunized child, fever, sore throat, a grey colored membrane
over both tonsils, neck swelling
Exercise 28.2:
Diphtheria: Heat-fixed smear of the colonies obtained on blood agar is provided. Perform the
Gram stain, focus under the microscope, record your observations and interpret the results
OR
Peritonsillar abscess: Pus sample is aspirated from peritonsillar abscess in a child. A heat-fixed
smear prepared from the colonies isolated on blood agar is provided. Perform the Gram stain,
focus under the microscope, record your observations and interpret the results
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29. Laboratory diagnosis of Lower respiratory tract infections (LRTI)
 Case based exercises with prepared smear for reporting by individual students
 Role play for instructions to be given to patient for sputum collection
 Small group case discussion for key difference in clinical presentation, etiological agent,
choice of sample collection, microbiological investigation for Lower Respiratory tract
infection and interpretation of Laboratory reports.
Exercise 29.1: Pneumococcal pneumonia

Clinical Case: A case of acute fever and cough with expectoration
Key points for case scenario: Adult, alcoholic, perspiration, cough, headache, chills and
shaking, Sputum yellowish green, but not foul smelling, Gram stain was performed and
following morphologies were seen
Exercise 29.2: Pulmonary tuberculosis

Clinical Case: A case of longstanding productive cough with weight loss
Key points for case scenario: Young adult, few weeks duration, cough with expectoration, low
grade fever, malaise, some weight loss, Not responding to a course of antibiotics
Exercise 29.3: Aspergilloma

Clinical Case: A case of old treated tuberculosis presenting with hemoptysis
Key points for case scenario: Adult,old treated pulmonary tuberculosis, breathlessness, cough
with blood-stained sputum, chest radiograph – intra-cavitary mass with air crescent sign
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BLOCK VII: Genitourinary & Sexually
transmitted infections
30. Laboratory diagnosis of Genitourinary and Sexually transmitted diseases

(Urethritis, Genital ulcers)
Suggested Teaching-Learning methods-

 Small group discussions based on case scenario for identification of pathogen and
interpretation of laboratory report
 Video demonstration of technique of sample collection in diagnosis of
genitourinary&sexually transmitted infections.
 Role play for counseling a patient about preventive aspects of STI.
Exercise 30.1: Primary syphilis

Clinical Case: A case of painless genital ulcer
Key points for case scenario: Young adult male, exposure to sex worker, genital lesion -
painless, circumscribed, indurated and superficially ulcerated
Exercise 30.2: Herpes genitalis

Clinical Case: A case of painful genital lesions
Key points for case scenario: Adult male, immunocompromised, multiple painful vesicular
lesions over penis and bilateral painful inguinal lymphadenopathy
Exercise 30.3: Trichomoniasis

Clinical Case: A woman with vaginal itching and discharge
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Key points for case scenario: Young adult female, burning micturition, severe vaginal itching,
wet mount preparation of vaginal secretion, motile organisms measuring approximately 12 um in
diameter
31. Laboratory diagnosis of Urinary tract infections (UTI)

 Small group discussions based on case scenario for identification of pathogen and
interpretation of laboratory report
 Demonstration of technique of sample collection in a catheterized patient
 Role play for instructions to be given when asking patient for midstream urine
sample collection.
 Demonstration of urinary tract pathogens like E. coli, Proteus spp,
Enterococcus Spp (Gram stain, Colony characteristics, Identification tests etc),
method of colony counting
Exercise 31.1: Urinary tract infection
Clinical Case: A case of fever and burning micturition
Key points for case scenario: young woman, increased frequency of micturition, burning
micturition, fever, urine culture
259

CBME Microbiology Practical Record Apurba Sastry

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CBME Microbiology Practical Record Apurba Sastry

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GSL MEDICAL COLLEGE

Fill the particulars given below

It is certified that, this is a bonafide record of the practical exercises of

Microbiology done byMiss / Mr your name

entered in this record book and his/her progress is satisfactory.

Signature of the Teacher in-charge Professor and Head

Name: Department of Microbiology

General Microbiology, Immunology &

 Describe the structure and functions of Microbiology laboratory

Section Diagnostic tests done

Bacteriology Staining & microscopy, culture, biochemical tests, serology tests,

Immunology Rapid immunochromatography tetsts, ELISA, FIA, CLIA, western blot

Mycology Staining & microscopy, culture, biochemical tests. They help to

Koch’s Postulate Demonstration

5. Specific antibodies to the disease Variable antibody response seen

3. Discuss the significance of proper hand hygiene

4. Enumerate the diseases that can be transmitted through contaminated hands

Scenario Biosafety level

BSC class I 1- provides Protection to the environment and the laboratory

BSC class II 2 - provides protection additionally to samples handled inside the

1) Enumerate different types of microscopes used in diagnostic microbiology laboratory

2) Describe the principle and uses of various microscopes

Case scenario Microscope Magnification Method

Stool examination for Light 400x Saline mount

Urine examination for pus Light 400x Direct mount

Bacterial examination of CSF Light 1000x Gram stain

Bacterial examination of Dark field - -

Best resolution 0.2 microns 0.5 nanometers

Radiation source Visible light Electron beam

Medium of travel Air Vacuum

Specimen mount Glass slide Copper grid

Type of lens Glass Electromagnet

Highest practical 1000 – 1500 100,000

Sl.No. Student’s performance Score#

1 Comes prepared with requisite prior knowledge 1 2 3

Date : Faculty Name & Signature

# Mark as 1, 2, 3 for ‘Not satisfactory’, ‘satisfactory’ & ‘Very Good’ respectively

Specific Learning Objectives:

At the end the session, the students shall be able to,

1) Suggest suitable clinical samples to be collected in the following infections/diseases

Type of infection/disease Suitable Clinical sample to be collected

Conjunctivitis Conjunctival swabs

Otitis media Middle ear aspirate

Oral and dental infections Swabs, aspirates, tissue specimens

Anaerobic – tissue specimens, aspirates

Septicemia Pair of 8 – 10 ml blood samples from different sites

Diarrhea, Dysentery, Food poisoning Stool, rectal swab, food sample

Urinary tract infections Urine – MSU, suprapubic aspirate, from catheter,

Diseases requiring serological/immunological 2ml Blood in vacutainer

Anaerobic culture media Anaerobic culture methods

Robertson’s cooked meat broth 1. Evacuation and replacement

4. What are the criteria for sample rejection?

5.What are the general principles to be followed for specimen collection?

See page No.225 in this record and label

(Source: Center for Disease Control, Atlanta, USA)

7. Write the general guidelines for specimen transportation

8. What are the important and essential components of specimen label?

11. Write the methods used for bacterial identification

1. Colony morphology properties

12. Write in brief on professional conduct pertaining to the clinical samples

Essential background knowledge required is :

Exercise 2.2: Demonstrate respect for patient samples

Specific Learning Objectives:

1 Comes prepared with requisite prior knowledge 1 2 3