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MICROBIOLOGY
Nanette Ramilo-Cruz, MD, DPAFP
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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
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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
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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
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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
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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
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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
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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%
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Microbiology
� The study of organisms too small to be seenwithout magnification
± bacteria
± viruses
± fungi
± protozoa
± helminths (worms) ± algae
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Branches of study within
microbiology� Immunology
� Public health microbiology & epidemiology
� Food, dairy and aquatic microbiology� Biotechnology
� Genetic engineering & recombinant DNA
technology
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Microbes are involved in
� nutrient production & energy flow
� decomposition
� production of foods, drugs & vaccines� bioremediation
� causing disease
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Impact of pathogens
� Nearly 2,000 different microbes cause
diseases
� 10 B infections/year worldwide
� 13 M deaths from infections/year worldwide
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Spontaneous generation
Early belief that some forms of life
could arise from vital forces present innonliving or decomposing matter.
(flies from manure, etc)
History
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Antonie van Leeuwenhoek
� First to observe living
microbes
� his single-lens
magnified up to 300X
(1632-1723)
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Otto Muller� Organized bacteria
into genera and
species
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Friedrich Henle� Microorganisms
were responsible
for causing diseases
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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.
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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)
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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.
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Robert Hooke
� Cell describe the
basic unit of life
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Louis Pasteur� Showed microbes caused
fermentation & spoilage
� Disproved spontaneousgeneration of m.o.
� Developed aseptic
techniques.
� Developed a rabies vaccine.
(1822-1895)
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Joseph Lister
� 5 April 1827 10
February 1912
� Pioneer of
antiseptic surgery
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Paul Ehrlich
� 14 March 1854 20
August 1915
� hematology,immunology and
chemotherapy
� Cured Syphilis
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Edward Jenner
� 17 May 1749 26
January 1823
� Pioneer of Smallpoxvaccine
� Father of
Immunology
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Alexander Fleming
� 6 August 1881 11
March 1955
� 1923: lysozyme� 1928: Penicillin
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Reasons for Studying Microbes
� Understand the disease they cause
� Ways to control them
� Ways to prevent the occurrence of
disease
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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
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Must know
Interaction results to:
� transient colonization
� long term symbiotic relationship
� Disease
Factors:
� Virulence
� Site of exposure
� Hosts ability to respond
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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
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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
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Taxonomy - system for organizing,
classifying & naming living things� Domain - Archaea, Bacteria &
Eukarya
� Kingdom - 5� Phylum or Division
� Class
� Order
� Family� Genus
� species
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3 domains
� Eubacteria -true bacteria, peptidoglycan
� Archaea odd bacteria that live in extreme
environments, high salt, heat, etc� Eukarya- have a nucleus, & organelles
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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)
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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
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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
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Virus
� Smallest infectious particle
� Obligate intracellular parasites
� No cellular structure� Consists of either DNA or RNA only
� Exceptions: Prions and Mimivirus
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Fungi
� Eukaryotic
� Types:
- Yeast:- Mold
- Dimorphic: Histoplasma, Blastomyces,
Coccidioides
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Diagnostic Microbiology
Nanette Ramilo-Cruz, MD, DPAFP
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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
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Diagnostic Medical Microbiology
� Identification of the causative microorganism
� Susceptibility testing
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Common Biological Specimens
� Blood/serum
� Sputum/bronchial washings
� Exudates/transudates� Urine and other body fluids
� Feces
� Swabs of tissue samples
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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
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� Collection of specimens
Proper method of collection
Proper labeling of specimens� Perform the diagnostic test
� Feedback information to the Physician
Diagnostic Medical Microbiology
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5 Laboratory Techniques
� Microscopic method
� In-vitro cultivation and Identification
� Detection of Microbial Enzymes� Detection of Microbial Genetic
material
� Serologic Diagnosis (Antibody andantigen)
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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)
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1. Microscopic Method
- For initial detection of microbes
- Identification of these microbes
- Methods:� Light microscopy
� Darkfield Microscopy
� Phase-contrast Microscopy� Flourescent Microscopy
� Electron Microscopy
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� Direct examination
� Differential Stain- Gram Strain
- Wright-Giemsa
- Ziehl-neelsen stain- Kinyoun stain
- 10% KOH
- India ink
- Wet mount.
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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
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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.
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Basic Steps in Gram Stain
� Heat-fix bacterial smear
� Apply the Crystal Violet
� Apply Grams Iodine� Rapid decolorization with Alcohol/ acetone
� Counterstain with Safranin
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Gram positive Bacteria
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Gram Negative Bacteria
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Acid Fast
� Physical property of some bacteria referring to
their resistance to decolorization by acids
during staining procedures.
� Ziehl-Neelsen Stain
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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.
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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
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Acid Fast Bacteria
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Endospores
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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
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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
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Negative Stain
� Uses Nigrosin
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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
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Culture of Bacillus anthracis
P H dli f Mi bi l
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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
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Types of Culture Media
� Enriched Non-selective media
� Selective Media
� Differential media� Specialized media
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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
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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
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B i I i P H dli f
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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
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� 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