South Asian Institute

of

Technology and Medicine

Malabe

Sri Lanka

Faculty of Medicine

HANDBOOK

FOR STUDENTS

IN

MEDICAL MICROBIOLOGY

and

IMMUNOLOGY

2014

2

CONTENTS

1. Introduction

2. The staff in the department

3. The time table

4. The course content and topics

5. General objectives of the course

6. Specific objectives in

6.1 General microbiology

6.2 immunology

6.3 systematic bacteriology

6.4 virology

6.5 mycology

6.6 antibiotics

7. The teaching learning methods

7.1 Lectures

7.2 Practical sessions and demonstrations

7.3 Small group discussions

7.4 Tutorials

8. Assessments

8.1 Continuous summative and formative assessments

8.1.1 Control tests

8.1.2 Credit tests

8.2 Final assessment in microbiology and immunology

9. Recommended reading

APPENDICES

1. Gram Stain

2. Acid Fast stain

3. Gram stain assessment

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1. Introduction

Welcome to the Department of Microbiology and to the course in

Microbiology.

Microbiology is the study of microscopic organisms. This course deals with

all aspects of microorganisms (bacteria, viruses and fungi), that cause

disease in humans, and those that have the potential to cause disease.

The learning of Microbiology should go hand in hand with your clinical

work in the wards. You should be able to relate the knowledge you learn in

the subject to cases of infectious diseases that you see and discuss in the

wards. In this way the subject becomes more interesting and relevant to

your ward work and also when you complete this course and move on to

become a medical doctor.

2. The staff in the department

Academic staff

Professors/ Senior Lecturers:

Nelun de Silva MBBS (Patna), Dip Micro(Col), MD Micro (Col)

Anura Weerasinghe -MBBS, MD, FRCP, DCH, DTM&H, FCCP, PhD

Nalaka Kanakaratne MD (Rus) MSc. (Kelaniya) PhD (Pera)

Lecturers:

Dr. Vindya Perera – BVSc (Peradeniya)

Ms. J. Lohitharajah -BSc (Human Biology)(Hon) – on study leave

3. The time table

You will be with us learning Microbiology during semesters IV and V in

the medical curriculum. Each of you will have about 8 hours of contact time

per week with the staff in our department during the semester 1V and 4

hours in semester V, learning Microbiology. These are distributed among

the various teaching learning activities, which are described in detail below.

This should not mean that you have to limit yourself to learning

Microbiology during these hours. The contact time you have with us is for

the purpose of directing your learning and to facilitate your learning

process. Therefore you will need to devote many more hours where you

will learn on your own or in small groups.

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At a glance the hours that are set aside for Microbiology in a week are as

follows:

SEMESTER 1V

6hours Lectures in Microbiology and Immunology

2 hours Small group discussions

Hands on Practical sessions

Practical demonstrations

Tutorials, Case studies

SEMESTER V

2 hours Lectures in Microbiology - on line and face

to face

2 hours Small group discussions

Practical demonstrations

Tutorials, Case studies

4. The course content and topics

Introduction to bacteriology.

Classification & Structure of bacteria.

Disinfection & Sterilization.

Pathogenesis & virulence of bacteria.

Detection, Culture & identification of Bacteria.

Bacterial genetics & bacteriophages, Molecular Biology &

genetic engineering.

Normal flora

Introduction to Immunology, Immune system,

Innate (non-specific) Immunity, Acquired (adaptive,

specific) immunity, Cells & tissues involved in the immune

system, Antigens, Innate immunity- Mechanical barriers,

Cells involved in innate immunity.

Innate immunity - Humoral aspects including Complement.

Adaptive immunity, Immunoglobulins & Humoral immunity.

Cell mediated immunity & Hypersensitivity.

Methods of acquiring Specific immunity.

Auto immunity & Immune tolerance.

Immuno deficiency & Transplantation. Immunity to bacterial, viral & fungal infections.

Immunization.

Staphylococci/ Streptococci / enterococci.

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Gram +ve bacilli ( Corynebacteria & Bacillus sp.).

Gram - ve cocci -- Neisseria.

Small Gram -ve bacilli(Haemophilus etc.)

Salmonella/ Shigella/ E.coli.

Pseudomonas/ vibrio / Aeromonas / Plesiomonas

Anaerobes -- Clostridia / Bacteroides.

Campylobacter/ Helicobacter.

Mycoplasma/ Rickettsiae/Chlamydiae.

Mycobacteria / Norcardia / Actinomycetes.

Spirochaetes.

Antibiotics / Antimicrobial therapy.

Introduction to Virology.

Viruses of the Respiratory tract

Hepatitis.

Herpes group of viruses.

pox viruses/ parvovirus/ papilloma viruses.

Mumps / Measles/ Rubella.

Rabies.

HIV .

Diarrhoeagenic viruses.

Entero & Arboviruses.

Mycology.

Collection & transport of specimens

UTI.

Upper respiratory infections.

Bone & joint infections.

PUO / Sepsis / Septic shock. Congenital, perinatal infections & blood borne infections. Infections of CNS & lower respiratory tract infections.

Abdominal infections, Gastroenteritis & food poisoning. Hospital acquired infections and infection control.

Eye & Ear Infections.

Bone and joint infections. Sexually Transmitted Diseases.

Skin Infections.

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5. General objectives of the course At the end of the teaching-learning course in Medical Microbiology the student should be able to:

- List the generic names of common microorganisms (bacteria, viruses & fungi), which cause disease in humans. - Select the ones that are frequently encountered and prioritise

them in order of importance - Describe their habitat and routes of transmission and explain the

pathogenesis of infections caused by them - Relate the clinical signs and symptoms of these diseases to the

underlying pathogenesis - Explain the mechanism of action, identify the spectrum of activity

of anti microbial agents and describe the resistance mechanisms of microorganisms against antimicrobials.

- Select appropriate antimicrobial agents that can be used in treatment and prophylaxis, being aware of on guidelines for empiric therapy, culture and antibiotic susceptibility test (ABST) reports, limitations (cost, availability, patient factors, etc..)

- Explain measures for the prevention and control of these diseases. - Advice on immunisation procedures for bacterial & viral diseases - Recognise the basic microscopic features of the common bacterial

pathogens and fungi and identify them in direct mounts, Gram and acid fast stained smears.

- Assist in the diagnosis of infectious diseases by o Identifying the microbiological tests available for the

diagnosis of these infections o Selecting the appropriate test according to the duration

of the illness o Advising on collection and transport of relevant

microbiological specimens - Define and list the antiseptics, disinfectants and sterilising agents,

explain their mode of action and select the appropriate ones for use in patient care and in the laboratory

- Describe the immunological mechanisms that come into play when a human host is exposed to a pathogen

- Interpret these mechanisms when selecting tests for immunological diagnosis in the context of available tests in the country

- Perform Gram stains of prepared smears of cultures or clinical specimens and identify as far as possible the microorganisms there in by their microscopic morphology and staining characteristics.

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6. Specific objectives

6.1 SPECIFIC OBJECTIVES IN GENERAL MICROBIOLOGY

At the end of each session of teaching – learning on general

microbiology, the students should be able to:

1. List the generic names of common microorganisms (Bacteria,

Viruses & Fungi), which cause disease in humans.

2. Recognize the basic microscopic features of the common

bacterial pathogens and Fungi and identify them in direct mounts,

Gram and acid fast smears.

3. Select the microbes that are encountered in this country and

prioritize them in order of importance.

3.1 Describe the habitat and routes of transmission and explain

the pathogenesis of infections caused by these.

3.2 Relate the clinical signs and symptoms of these diseases to

the underlying pathogenesis.

3.3 List microbiological tests available for the diagnosis of these

infectious diseases.

3.4 Select appropriate tests according to the duration of the

illness.

3.5 Advise on and be able to collect and transport relevant

microbiological specimens.

3.6 Select appropriate antimicrobial agents that can be used in

treatment and prophylaxis, being aware of

the guidelines for empiric therapy, culture and antibiotic

susceptibility test (ABST) reports and limitations( cost, availability,

patient factors, etc..)

3.7 Explain the measures for the prevention and control of

these diseases.

4. Explain the mechanism of action, identify the spectrum of

activity of antimicrobial agents and describe the resistance

mechanisms of microorganisms against antimicrobials.

5. Advise on appropriate immunization procedures for the bacterial

and viral diseases.

6. Define and list the antiseptics, disinfectants and sterilizing

agents, explain their mode of action and select the appropriate

ones for patient care and in the laboratory.

7. Describe the normal and exaggerated immunological responses

of a human host when exposed to a pathogen / foreign antigen/

self antigens.

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7.1 Interpret these responses when selecting tests for

immunological diagnosis of infectious diseases, being aware of the

tests available in the country.

8. Perform the Gram stain in clinical specimens and identify

bacteria by their staining character and microscopic morphology.

6.2 SPECIFIC OBJECTIVES IN SYSTEMATIC BACTERIOLOGY

At the end of each session of teaching – learning on Systematic

Bacteriology, the students should be able to:

1. List the bacterial species that cause important and

common infective diseases.

2. Define the characteristics of the genera to which these

bacteria belong.

3. Identify their natural source, habitat and growth

requirements for artificial culture in the laboratory

4. Identify them by their microscopic morphology, and

colony appearance

5. List the infections or clinical syndromes they cause in

humans.

6. Explain the pathogenesis of infections they cause.

7. Advise staff and patients regarding collection and

transport of clinical specimens for the microbiological

diagnosis of infective diseases.

8. Interpret reports of microbiological investigations done

in these infective diseases

9. List front line and second line antibiotics effective against

major pathogenic bacteria and select appropriate

antibacterial agents for the treatment of infections

caused by them.

10. Explain the epidemiology of such infections and update

themselves on the prevalence of infective diseases in the

community.

11. Formulate methods to prevent and control infections in

the community and in the hospital, caused by the

bacteria based on their habitat and modes of spread.

12. Perform direct smears of clinical specimens / broth or

solid cultures, stain appropriately and identify the

bacteria as far as possible.

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6.3 SPECIFIC OBJECTIVES FOR SYSTEMATIC VIROLOGY.

At the end of each session of teaching – learning on

systematic virology, the students should be able to:

1. State some historical facts regarding the virus or the group of

viruses or the discovery of it.

2. Identify the common characteristics of the family and the

genus to which the virus belongs.

3. Identify the important and pathogenic serotypes /strains /

variants of the genus.

4. Explain the morphology and common characteristics of the

virus.

5. Explain the methods of transmission of the virus to the

human host, and the natural hosts and vectors (if any).

6. List the common diseases and syndromes caused by the virus.

7. Recognize and describe briefly the clinical features of

common diseases and syndromes caused by the virus.

8. Explain the pathogenesis and outcome of such viral

infections.

9. Choose the appropriate laboratory diagnostic methods

available for detection of the viral infection.

10. Instruct how the appropriate clinical specimens are collected

and transported for laboratory diagnosis of viral infection.

11. Interpret laboratory reports for viral infections.

12. Prescribe appropriate anti viral drugs (if any) for such viral

infections.

13. Describe briefly the epidemiology (i.e. the prevalence,

distribution, at risk population etc) of such infections.

14. Explain the appropriate methods of prevention and control of

viral infections in the community.

6.4 SPECIFIC OBJECTIVES FOR MYCOLOGY

At the end of each session of teaching – learning on Mycology,

the students should be able to:

1. List the aetiological agents and identify the clinical features

and recognize common superficial, subcutaneous and

systemic fungal infections. Eg. Taenia, kerion, pityriasis

versicolor, piedra, onychomycosis, candidasis,

chromomycosis, mycetoma and rhinosporidiosis,

aspergillosis and cryptoccosis.

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2. Describe their pathogenesis.

3. Advise on collection and transport of specimens for their

laboratory diagnosis.

4. Treat and advise on prevention (if any) of superficial fungal

infections.

6.5 SPECIFIC OBJECTIVES FOR ANTIMICROBIAL AGENTS

At the end of each session of teaching – learning on Antibiotics,

the students should be able to:

1. Recall some interesting facts in the history of development of

antimicrobials.

2. Explain the mechanisms of action of antimicrobials.

3. Describe the origin and types of drug resistance by microbes.

4. Identify the clinical settings where antimicrobial agents are used

for empiric and definitive therapy.

5. Identify the clinical conditions where antimicrobials are used for

prophylaxis.

6. Outline the tests that are done in the laboratory, to guide

clinicians on antimicrobial therapy.

7. Describe the development of antimicrobial drug resistance by

microbes and its clinical implications.

8. List the generic names, describe the mechanism of action,

spectrum of activity, adverse effects of commonly used

antimicrobials in each group e.g. penicillins, aminoglycosides,

cephalosporins, azoles etc.

9. Identify the drug/s of choice and schedules in the treatment of

common infective diseases such as Tuberculosis, Leprosy,

Typhoid, Infective endocarditis, UTI, pneumonia, gastro-

enteritis, meningitis & encephalitis, taenia infections, herpes

infections etc.

10. Interpret antibiotic susceptibility test reports issued by the

laboratory.

11. Select an appropriate antimicrobial to treat common infective

diseases.

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6.6 SPECIFIC OBJECTIVES FOR IMMUNOLOGY 1. Basic Immune mechanism

a. Innate Immunity 1. define the term "innate immunity" 2. describe the innate immune mechanisms found in the body:

cells and molecules 3. define the term "acquired immunity" and describe how it

differs from innate immunity b. Acquired immunity

1. list the types of acquired immunity 2. define the term "antigen' 3. list the antigen-presenting cells (APCs) and explain how APCs

present the antigens on their cell surfaces 4. state the sites of location of lymphocytes 5. define the terms "cell-mediated immunity" and "humoral

immunity" and state the type of lymphocyte that promotes each of them

6. give the sites of pre-processing of the T and B lymphocytes 7. explain the mechanism of activation of lymphocytes by

specific antigens to form clones 8. explain the mechanism of specificity of the T and B

lymphocyte clones 9. explain how antigens are presented to the T and B

lymphocytes a. Humoral immunity

1. describe the series of events which take place from the time a B lymphocyte encounters an antigen up to the formation of antibodies

2. explain the difference between the primary and the secondary antibody response

3. recognise that antibodies are gamma globulins which are called immunoglobulins

4. state the different types of immunoglobulins and describe their functions b. Cell-mediated immunity

1. describe the events taking place when a T lymphocyte is exposed to an appropriate antigen

2. recognise that T cell markers (surface receptor proteins) are firmly bound to the cell surface

3. list the types of T cells and describe the functions of each of them c. Introduction to applied immunology

1. Hypersensitivity 2. Autoimmunity 3. Immunodeficiency

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7 The teaching learning activities (TLA)

7.1 Lectures

The permanent academic staff of the department will conduct the

lectures. This is the main TLA in this course where the core

knowledge in Microbiology will be learnt. The schedule of lectures

together with the dates will be displayed on the notice board at the

beginning of semester 1V and V. It would be good if you can come

prepared for these lectures in which case you will be able to follow

the lecture very clearly and make this TLA a productive learning

process.

The teachers will use power point presentations to facilitate

learning during the lecture. Handouts of the slides will be provided

most of the time before the lectures. Some clinical Microbiology

lectures will be available on line on EDU 2.0 in semester V. Students

need to register to access these online lectures.

The topics are as follows:

SEMESTER 1V

Basic concepts in immunology , Antigens.

Antibodies/ Immunoglobulins (part 1).

Antibodies/ immunoglobulins (part 2).CD-antigens. Antigen-

recognizing receptors of B- and T-lymphocytes

Major Histocompatibility Complex (MHC/ HLA).

MHC-dependent restriction and regulation of immune response

Cytokines. Functional variants of CD4 T-cells.

Induction of immune response immunogenesis

Effector stage of immune response: humoral immunity.

Effector stage of immune response: cellular immunity

Antiviral immunity

Structure of bacteria, bacterial genetics

Introduction to Bacteria , pathogenecity and virulence of

microorganisms: general conceptions & toxins

Introduction to viruses/ Antiviral agents, Sterilization & disinfection

Systematic bacteriology, mycology & antifungal agents

Antimicrobials and antimicrobial chemotherapy

SEMSTER V

Systematic virology and Clinical microbiology

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7.2 Practical sessions

The practical sessions in the semester 1V will train you to be

competent in performing the Gram stain (appendix 1).

You will be working in pairs or threes doing the staining

procedure, after which you will individually focus the stained

smear under the microscope and observe the microscopic

morphology and staining character of the bacteria in the smear.

The acid fast stain (Ziehl Neelson) procedure will be demonstrated

to you in this semester. (appendix 2)

7.3 Small group discussions

The students are exposed to this type of teaching learning activity

during the scheduled time for practical or lectures in Microbiology.

The batch will be divided into small groups of 10-12 students in

each group and the topic to be discussed will be given as questions

or clinical scenarios. At the end of the allocated time

lecturer/tutor will facilitate the discussion.

7.4 Tutorials

Some scheduled practical hours in Microbiology are utilized for

tutorials. Students are given the topic or questions a few days

before and active discussion carried out during the tutorial.

8. Assessments in Microbiology and Immunology

8.1. Continuous summative and formative assessments

8.1.1 Control Tests

These are purely formative assessments to ensure that students

are up-to-date with their course work These will be conducted on

different topics as scheduled tests or at the beginning of a lecture

on previous lecture topics. The objective of these tests is to give

feedback to students on how well they are progressing in their

course work in Immunology and Microbiology.

The format of these tests will be true false type of MCQ's,

Objective structured Practical examination (OSPE)or short answer

questions.

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8.1.2 Credit tests

Credit tests are conducted in each semester. These assessments

are formative as well as summative in nature. The total mark

obtained in credit tests will contribute to 20% of the total mark in

the final examination in Microbiology and Immunology.

The format of the credit tests will be any one of these methods

with a duration of approximately 30 to 60 mins. :

- A practical test to ascertain the capability of the student to

perform a Gram stain on a smear. They will be assessed on the

quality of the Gram stain. Focusing capability, observations and

interpretation.

- True false type of MCQ's, single best response (SBA) MCQ’s or

extended matching items (EMI)

- Short answer questions (SAQ)

- Objective structured Practical examination (OSPE)

8.2. Final assessment in microbiology and immunology at the end

of Vth

semester.

The theory component will comprise of :

a. Four Structured Essay Questions (SEQ) or 12 short answer

questions (SAQ’s) to be answered in 2 hours.

b. Twelve true false MCQ’s , two SBA MCQ’s and three EMI’s to

be answered in one hour.

c. Twenty Objective structured Practical examination (OSPE)

where power point projections are displayed with questions and

students are expected to answer in one hour.

d. Viva voce will be conducted by 4-5 panels of examiners.

Each panel will have two examiners and may include external

examiners as well. Each student will be examined by a single panel

for 8-10 mins.

Compilation of the final mark as a percentage – main examination

SEQ/SAQ 30

MCQ 20

OSPE 20

viva voce 10

Credit tests at end of semester 1V & V 20

Total 100

Pass mark : 50%

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Students obtaining less than 50% in the total mark has to repeat

all components of the examination

To be eligible for distinction a student should obtain at least 75%

in the total mark and performed equally well in all components of

the examination.

Compilation of the final mark as a percentage – Repeat examination

SEQ/SAQ 40

MCQ 25

OSPE 25

Viva voce 10

Total 100

9. Recommended Reading

1. Medical microbiology by D. Greenwood, R. Slack, J. Peutherer.-Churchill

Livingstone.

2. Medical Microbiology by Brooks G.F. et al. Jawetz, Melnick,

Adelberg's . 21st

ed. Appleton & Lange, 1998.

3. Medical Microbiology by Mims C. A., Playfair J. H. L., Roitt I., Wakelin

D., Williams R., Anderson R. M... Mosby. 1993.

4. Immunology by Roitt I., Brostoff J., Male D. 5th

ed. Mosby. 1998.

5. Basic immunology by Sharon J.. Williams & Wilkins. 1998.

6. Immunology by Weir D. M., J. S. Churchill Livingston. 8th

ed. 1997.

APPENDICES

APPENDIX 1: PRESTON & MORELL'S MODIFICATION OF GRAM'S STAIN

1. Apply Ammonium Oxalate-Crystal Violet stain for 1/2 minute.

2. Wash thoroughly with water.

3. Apply Lugol's Iodine solution for 1/2 minute.

4. Wash thoroughly with water.

5. Apply Iodine-Acetone solution for 1/2 minute.

6. Wash thoroughly with water.

7. Counter stain with dilute Carbol Fuchsin for 1/2 minute.

8. Wash thoroughly with water.

The whole smear must be flooded with each reagent and the previous

reagent must be completely washed at each stage. Care must also be taken

not to waste stains by over flooding the slide with the stain.

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APPENDIX 2: ZIEHL-NEELSEN'S METHOD FOR ACID-FAST BACILLI

1. Flood the slide with Strong Carbol Fuchsin and heat, until steam rises.

(do not boil).

Allow the preparation to stain for 5 minutes, Heat being applied at

intervals to keep the stain hot. The stain must not be allowed to

evaporate and dry on the slide, if necessary pour more carbol fuchsin

to keep the whole slide covered with stain.

2. Wash the slide thoroughly under running water.

3. Decolourize in 3% acid alcohol by pouring the solution onto the smear.

Keep till the red colour of the smear is changed to light pink, Wash the

slide in water. The object of the washing is to remove the compound

acid with stain. The decolourization is finished when, after washing the

slide, the film is very faintly pink.

4. Wash the slide well in water.

5. Counterstain with methylene blue for 2 minutes

7. Wash the slide

APPENDIX 3: CHECK LIST FOR PERFORMANCE OF GRAM STAIN

1. Observes the correct side of the slide on which the

smear has been made

2. Pours ammonium oxalate/crystal violet - just enough to

cover smear

3. Adjusts the rack if needed

4. Opens tap / adjusts water stream

5. Times each step

6. Washes adequately /Pours water on stain before

discarding

7. Pours Lugols iodine & times the procedure

8. Washes adequately

9. Pours iodine acetone & times precisely

10 Washes well

11 Counterstains with dilute carbol fuchshin & washes well

12 Dries on filter paper by pressing