copy lec june 6 2011 microbiology

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MICROBIOLOGY

Nanette Ramilo-Cruz, MD, DPAFP



BASIC COURSE DESCRIPTION:

Basic knowledge and skills:a. Classify of bacteria, viruses and fungi of medical and

public health importance

1.characteristics for isolation and identification

2. capacity to produce disease

3. distribution and mode of transmission

4. host response

5. prevention and control.

b. Identify different diagnostic tests for common

infectious diseases

c. Interpretation/clinical correlation of laboratory

results



RECOMMEMDED TEXTBOOKS & REFERENCES

� Jawetz, Menick & Adelbergs Medical

Microbiology, 23nd edition

� Medical Microbiology. Murray, et al. 6th

ed.2009

� Foundation in Microbiology, K athleen Park

Talaro , 3rd edition, 1999

� Microbiology Reviewers



DATE TOPICS

June 6 Mon 1-3pm 1. Intro to Microbiology

2.Methods used in Diagnostic Microbiology

June 9 Thurs 1-5pm 1. Host-parasite interaction

2. Principles of disease transmission

June 13 Mon 1-3pm Part I:Review of the Immune System Antigens, Antibodies, Complements and

MHC

June 16 Thurs 1-5pm Part II: Review of the Immune System Antigens, Antibodies, Complements and

MHC

June 20 Mon 1-3pm Immune Response

June 23 Thurs 1-5pm 1. Hypersensitivity, Tolerance and Autoimmunity

2. Immunological Tests Used in the Diagnosis of Disease

3. Review of Immunization

June 27 Mon 1-3pm Introduction to Bacteriology :

Structures of the Bacterial Cell

June 30 Thurs 1-5 pm Introduction to Bacteriology : 1.Bacterial Growth and Death2.Bacterial Genetics

July 4 Mon 1-3pm 1.Introduction to Bacteriology : Bacterial Metabolism

July 7 Thurs 1-5pm The Medically Important Bacteria : C orynebacteria, Staphylococci,

Streptococci ,Pneumococci and Enterococci

July 14 1ST LONG EXAM



July 18 Mon 1-

3pm

The Medically Important Bacteria :

Mycobacteriaceae, Actinomycetes, Aerobic Spore-forming Gram Positive Bacilli

July 21 Thurs 1-

5pm

Other Medically Important Bacteria : Enterobacteriacea , Vibrio,

C ampylobcater, Helicobacter

July 25 Mon 1-

3pm

1. Other Medically Important Bacteria : Neisseriaceae, Haemophilus,

Bordetella

July 28 Thurs 1-

5pm

The ANAEROBIC BACTERIA : 1. The Gram Positive bacilli , spore-forming

Anaerobes ( Clostridia) and the Anaerobic Gram positive cocci

2. The Gram negative anaerobes : Bacteroides ,Fusobacterium

Aug 1 Mon 1-

3pm

The Medically Important bacteria in food , water and milk

Aug 4 Thurs 1-

5pm

1. Pseudomonas and other nonfermenting Bacilli ( Acitenobacter,

Flavobacterium

2. Other Gram Negative bacilli causing infections in animals and humans :

Yersinia, Francisella, Pasteurella and Brucella

3.Other Pathogenic Microorganisms ( Legionella, Listeria monocytogenes,

Erysipelothrix rhusiooopathiae, Streptobacillus moniliformis,

C alymmatobacterium ( Donovania ) granulomatis, Bartonella bacilliformis.

Gardnerella vaginalis

Aug 8 Mon 1-

3pm

1. Spirochetes, Rickettsiae, Chlamydiae, Mycoplasma

Aug 11 Thurs 2ND LONG EXAM



Aug 15 Mon 1-3pm 1. Introduction to Virology and Diagnostic Virology

2. The DNA viruses I ( Adenoviruses, Pox Viruses and Human papilloma viruses)

Aug 22 Mon 1-3pm 2. herpes Virus

Sept 1 Thurs 1-5pm 1. The Hepatitidis ( Viruses causing infection of the liver)

2. The RNA viruses: Picornaviruses , Orthomyxoviruses and Paramyxoviruses

Sept 5 Mon 1-3pm 1. viruses, Reoviruses and other viral causes of Gastroenteristis

Sept 8 Thurs 1-5pm 1. Rhabdovirus, Togaviruses, Flaviviruses, Bunyaviruses and Arenaviruses

2. Rubella

3. Prions and other Emerging viral infections

Sept 12 Mon 1-3pm Retroviruses (HIV) and Oncogenic viruses

Sept 15 Thurs 1-

5pm

1. Introduction to Mycology

2. Superficial and Cutaneous mycoses3. Subcutaneous Mycoses

4. Systemic and Opportunistic Mycoses

Sept 19 Mon 1-3pm 1. Microbial control

Sept 22 Thurs 3rd LE

Sept 26 FINAL EXAM



Policies:

� Attendance checked every session

� Automatic Failure for those with

>20%absences

� Missed Long Exam: may take make-up examONLY if absences were deemed excused by the

Prefect.

� Present excuse letter approved by the prefectwithin 2 weeks from the first day of going back

to school.

� No Make-up for missed quizzes



Grading System:

� 3 Long Exams: 50

� Final Exam: 30

� Attendance: 10

� Participation/Quizzes 10

100%

� Final Exam Exemption: > or = 81% of Long ExamAverage

� Passing grade: 60%



Microbiology

� The study of organisms too small to be seenwithout magnification

± bacteria

± viruses

± fungi

± protozoa

± helminths (worms) ± algae



10

Branches of study within

microbiology� Immunology

� Public health microbiology & epidemiology

� Food, dairy and aquatic microbiology� Biotechnology

� Genetic engineering & recombinant DNA

technology



11

Microbes are involved in

� nutrient production & energy flow

� decomposition

� production of foods, drugs & vaccines� bioremediation

� causing disease



12

Impact of pathogens

� Nearly 2,000 different microbes cause

diseases

� 10 B infections/year worldwide

� 13 M deaths from infections/year worldwide



13



Spontaneous generation

Early belief that some forms of life

could arise from vital forces present innonliving or decomposing matter.

(flies from manure, etc)

History



15

Antonie van Leeuwenhoek

� First to observe living

microbes

� his single-lens

magnified up to 300X

(1632-1723)



Otto Muller� Organized bacteria

into genera and

species



Friedrich Henle� Microorganisms

were responsible

for causing diseases



Germ theory of disease

Many diseases are caused by the

growth of microbes in the body and not

by sins, bad character, or poverty, etc.



19

Heinrich Hermann Robert Koch

(11 December 1843 27 May 1910)

� Established a sequence

of experimental steps to

show that a specific m.o.causes a particular

disease (KOCHS

POSTULATE).

� Developed pure culturemethods.

� Identified cause of

anthrax, and TB.(1843-1910)



Kochs Postulate

� The microorganism must be found in abundance in

all organisms suffering from the disease, but should

not be found in healthy animals.

� The microorganism must be isolated from a diseasedorganism and grown in pure culture.

� The cultured microorganism should cause disease

when introduced into a healthy organism.

� The microorganism must be reisolated from theinoculated, diseased experimental host and

identified as being identical to the original specific

causative agent.



Robert Hooke

� Cell describe the

basic unit of life



22

Louis Pasteur� Showed microbes caused

fermentation & spoilage

� Disproved spontaneousgeneration of m.o.

� Developed aseptic

techniques.

� Developed a rabies vaccine.

(1822-1895)



Joseph Lister

� 5 April 1827 10

February 1912

� Pioneer of

antiseptic surgery



Paul Ehrlich

� 14 March 1854 20

August 1915

� hematology,immunology and

chemotherapy

� Cured Syphilis



Edward Jenner

� 17 May 1749 26

January 1823

� Pioneer of Smallpoxvaccine

� Father of

Immunology



Alexander Fleming

� 6 August 1881 11

March 1955

� 1923: lysozyme� 1928: Penicillin



Reasons for Studying Microbes

� Understand the disease they cause

� Ways to control them

� Ways to prevent the occurrence of

disease



Must know

� Common for a particular organism to produce

many diseases

� Few organisms can be classified as ALWAYSpathogenic

� Exogenous infections

� Endogenous infections� Interaction between microbes and host can

result in to different relationships



Must know

Interaction results to:

� transient colonization

� long term symbiotic relationship

� Disease

Factors:

� Virulence

� Site of exposure

� Hosts ability to respond



Characteristic

s

Eukaryotes Prokaryotes

Groups Algae, fungi,plants,

protozoa, animals

Bacteria

Nucleus Classic membrane No nuclear

membrane

Mitochondria Present Absent

Golgi bodies Present Absent

ER Present Absent

Ribosomes 80s 70s

C to lasmic Contains sterols No sterols



character

istic

Eukaryotes Prokaryotes

Reproduc

tion

Sexual and

asexual

Asexual (binary Fission

Respiration Via mitochondria Via Cytoplasmicmembrane

Cell Wall Present for fungi Contains proteins, lipids

and Peptidoglycan

Size >5 micrometer 0.5-3.0 micrometer

Chromos

ome

DNA diploid

genome

Single circular DNA

haploid



32



33

Taxonomy - system for organizing,

classifying & naming living things� Domain - Archaea, Bacteria &

Eukarya

� Kingdom - 5� Phylum or Division

� Class

� Order

� Family� Genus

� species



34

3 domains

� Eubacteria -true bacteria, peptidoglycan

� Archaea odd bacteria that live in extreme

environments, high salt, heat, etc� Eukarya- have a nucleus, & organelles



35

Naming micoorganisms

� Binomial (scientific) nomenclature

� Gives each microbe 2 names

± Genus - noun, always capitalized

± species - adjective, lowercase

� Both italicized or underlined

± Staphylococcus aureus (S. aureus)

± Bacillus subtilis (B. subtilis) ± Escherichia coli (E. coli)



Major Groups of Medically

Important Microorganisms� Bacteria: S. aureus, E. coli, V. cholera, S. pneumoniae

� Fungi: Malassezia species, C. albicans

� Virus: HIV, Hepatitis virus, Rotavirus, HPV

� Algae: Planktons, dinoflagellates

� Protozoa



Bacteria

� Prokaryotic

� Cell wall is complex: Gram (+) and gram (-)

� Typical Bacteria:

- Shape: rod, spheres, spirals

- Spacial arrangement: single. Chains, clusters

- have plasmids� Atypical Bacteria: Mycoplasma, Chlamydia,

Rickettsia



Virus

� Smallest infectious particle

� Obligate intracellular parasites

� No cellular structure� Consists of either DNA or RNA only

� Exceptions: Prions and Mimivirus



Fungi

� Eukaryotic

� Types:

- Yeast:- Mold

- Dimorphic: Histoplasma, Blastomyces,

Coccidioides



Diagnostic Microbiology

Nanette Ramilo-Cruz, MD, DPAFP



Objectives

� Discuss the functions of a Diagnostic MedicalMicrobiology

� Enumerate the common biological specimens

used in the diagnosis of infections diseases

� Discuss the common laboratory methodsused in the diagnosis of infectious diseases

� Discuss the proper method of collection,handling, storage of these biologicalspecimens



Diagnostic Medical Microbiology

� Identification of the causative microorganism

� Susceptibility testing



Common Biological Specimens

� Blood/serum

� Sputum/bronchial washings

� Exudates/transudates� Urine and other body fluids

� Feces

� Swabs of tissue samples



The physician should:

1.what laboratory examinations to request

2.Know when and how to take the specimens

3.Inform the Laboratory of the clinical

information and preliminary diagnosis

4.How to interpret the results



� Collection of specimens

Proper method of collection

Proper labeling of specimens� Perform the diagnostic test

� Feedback information to the Physician

Diagnostic Medical Microbiology



5 Laboratory Techniques

� Microscopic method

� In-vitro cultivation and Identification

� Detection of Microbial Enzymes� Detection of Microbial Genetic

material

� Serologic Diagnosis (Antibody andantigen)



Sensitivity and Specificity of Test

Results

� Need to know the reliability of a diagnostic

procedure

� Probability that a test will be positive in the

presence of a pathogen (all infected patients

are detected)

� Probability that a test will be negative if the

pathogen is not present (all positive patients

are infected)



1. Microscopic Method

- For initial detection of microbes

- Identification of these microbes

- Methods:� Light microscopy

� Darkfield Microscopy

� Phase-contrast Microscopy� Flourescent Microscopy

� Electron Microscopy



� Direct examination

� Differential Stain- Gram Strain

- Wright-Giemsa

- Ziehl-neelsen stain- Kinyoun stain

- 10% KOH

- India ink

- Wet mount.



Gram Staining

� 1882 Hans Christian Gram

� Differentiate bacterial species into two large

groups (Gram-positive and Gram-negative)based on the chemical and physical properties

of their cell walls



Gram Stain

� Gram-positive bacteria: thick mesh-like cell

wall made of peptidoglycan (50-90% of cell

wall), which stains purple

� Gram-negative bacteria: have a thinner layer

(10% of cell wall), which stains pink. Outer

membrane contains lipids, and is separated

from the cell wall by the periplasmic space.



Basic Steps in Gram Stain

� Heat-fix bacterial smear

� Apply the Crystal Violet

� Apply Grams Iodine� Rapid decolorization with Alcohol/ acetone

� Counterstain with Safranin



Gram positive Bacteria



Gram Negative Bacteria



Acid Fast

� Physical property of some bacteria referring to

their resistance to decolorization by acids

during staining procedures.

� Ziehl-Neelsen Stain



Ziehl-Neelsen Stain

� Cover with tissue paper

� Flood slide with carbolfuchsin, the primary

stain, for 2 minutes while heating with steam

or heating on hot plate.

� Remove paper cover, decolorize slide with a

mixture of hydrochloric acid and ethanol.

� Counter stain with methylene blue.



Notable Acid Fast Structures

� All Mycobacteria - M. tuberculosis, M. leprae,

M. smegmatis and atypical Mycobacterium

� Nocardia

� Head of sperm

� Bacterial spores

� Parasites likeC

ryptosporidium parvumIsospora and C yclospora cysts



Acid Fast Bacteria



Endospores



Potassium Hydroxide Test (KOH)

� Detects fungi

� Dissolve human cells. KOH denatures the

proteins in the human cell; only the fungal

cells remain to be seen under the microscope.

� Athlete's foot, fungal vaginitis and many other

fungal infections



KOH Test Procedure

� Take scraping from margin (not center) of lesion

� Place on clean slide

� Add 2-3 drops of 10% KOH in water

� Warm the slide (don't boil)

� Add cover slip

� Examine immediately under high dry

magnification with light microscope



Negative Stain

� Uses Nigrosin



2. Microbial Cultivation

� Method of multiplying microbial organisms by

letting them reproduce in predetermined

culture media under controlled laboratory

conditions

� Importance: Diagnostic Purposes

Prognosis of disease

� Using: Agar



Culture of Bacillus anthracis

P H dli f Mi bi l



Proper Handling of Microbial

Specimens

� Very Important!

� crucial for obtaining microbiological test

results that are both timely and clinically

relevant.

� Maximizes Cost-effectiveness of laboratory

test



Types of Culture Media

� Enriched Non-selective media

� Selective Media

� Differential media� Specialized media



3. Detection of Microbial Enzymes

� Single enzyme test:

- Catalase test

- oxidase test- Urease test

- Coagulase test

� Test on the presence of Metabolic pathways

4 D t ti f Mi bi l G ti



4. Detection of Microbial Genetic

Material

� High Sensitivity and specificity

� Safe

- Not require isolating the infectious agent- can be performed on a chemically fixed

sample

� Example: PCR, DNA Probes, ElectrophoreticAnalysis of DNA



B i I i P H dli f



Basic Issues in Proper Handling of

Specimens� Collection of Specimens� Important information includes:

* the specific site(s)

* whether the patient was receiving antibioticsprior to collection

* specific pathogens that are being sought

* the methods by which the specimen was

collected

* whether patient may be infected with

pathogens known to be dangerous to laboratory

staff.

B i I i P H dli f



� Transport of Specimens

� Storage of specimens

� Specimens that should not be refrigerated

include:

* blood--should be left at room temperature

or in an incubator at 5[degrees]C

* cerebrospinal fluid--transport at roomtemperature

* Neisseria species--transport rapidly to the

laboratory.

Basic Issues in Proper Handling of

Specimens

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