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 & Adelberg’s Medical

Microbiology, 23nd edition
• Medical Microbiology. Murray, et al. 6th ed.
2009
• Foundation in Microbiology, Kathleen Park
Talaro , 3rd edition, 1999
• Microbiology Reviewers
July 18 Mon 1- The Medically Important Bacteria :
3pm Mycobacteriaceae, Actinomycetes, Aerobic Spore-forming Gram Positive Bacilli
July 21 Thurs 1- Other Medically Important Bacteria : Enterobacteriacea , Vibrio,
5pm Campylobcater, Helicobacter
July 25 Mon 1- 1. Other Medically Important Bacteria : Neisseriaceae, Haemophilus,
3pm Bordetella
July 28 Thurs 1- The ANAEROBIC BACTERIA : 1. The Gram Positive bacilli , spore-forming
5pm Anaerobes ( Clostridia) and the Anaerobic Gram positive cocci
2. The Gram negative anaerobes : Bacteroides ,Fusobacterium
Aug 1 Mon 1- The Medically Important bacteria in food , water and milk
3pm
Aug 4 Thurs 1- 1. Pseudomonas and other nonfermenting Bacilli ( Acitenobacter,
5pm 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,
Calymmatobacterium ( Donovania ) granulomatis, Bartonella bacilliformis.
Gardnerella vaginalis
Policies:
• Attendance checked every session
• Automatic Failure for those with >20%absences
• Missed Long Exam: may take make-up exam ONLY
if absences were deemed excused by the Prefect.
• Present excuse letter approved by the prefect
within 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 Exam

Average
• Passing grade: 60%
 Microbiology
• The study of organisms too small to be seen
without magnification
– bacteria
– viruses
– fungi
– protozoa
– helminths (worms)
– algae
 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

11
 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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13
 History

Spontaneous generation

Early belief that some forms of life

could arise from vital forces present in
nonliving or decomposing matter.
(flies from manure, etc)
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
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.
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
(KOCH’S POSTULATE).
• Developed pure culture
methods.
• Identified cause of
anthrax, and TB.
(1843-1910)
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 Koch’s 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 diseased
organism and grown in pure culture.
• The cultured microorganism should cause disease when
introduced into a healthy organism.
• The microorganism must be reisolated from the
inoculated, diseased experimental host and identified as
being identical to the original specific causative agent.
Robert Hooke
• “Cell” – describe the
basic unit of life
 Louis Pasteur
• Showed microbes caused
fermentation & spoilage
• Disproved spontaneous
generation 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
Paul Ehrlich
• 14 March 1854 – 20
August 1915
• hematology,
immunology and
chemotherapy
• Cured Syphilis
Edward Jenner
• 17 May 1749 – 26
January 1823
• Pioneer of Smallpox
vaccine
• 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 ALWAYS
pathogenic
• 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
• Host’s ability to respond
Characteristic Eukaryotes Prokaryotes
s
Groups Algae, fungi,plants, Bacteria
protozoa, animals

Nucleus Classic membrane No nuclear

membrane
Mitochondria Present Absent
Golgi bodies Present Absent
ER Present Absent
Ribosomes 80s 70s
character Eukaryotes Prokaryotes
istic
ReproducSexual and Asexual (binary Fission
tion asexual
Respirati
Via mitochondria Via Cytoplasmic
on membrane
Cell Wall
Present for fungi Contains proteins, lipids
and Peptidoglycan
Size >5 micrometer 0.5-3.0 micrometer
Chromos DNA diploid Single circular DNA
ome genome haploid
32
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
 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 Medical
Microbiology
• Enumerate the common biological specimens
used in the diagnosis of infections diseases
• Discuss the common laboratory methods
used in the diagnosis of infectious diseases
• Discuss the proper method of collection,
handling, storage of these biological
specimens
 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
 Diagnostic Medical Microbiology
• Collection of specimens
Proper method of collection
Proper labeling of specimens
• Perform the diagnostic test
• Feedback information to the Physician
 5 Laboratory Techniques
• Microscopic method
• In-vitro cultivation and Identification
• Detection of Microbial Enzymes
• Detection of Microbial Genetic
material
• Serologic Diagnosis (Antibody and
antigen)
 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 Gram’s 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 like Cryptosporidium parvum
Isospora and Cyclospora cysts
Acid Fast Bacteria
 Schaeffer-Fulton Stain
• Isolate endospores
• Stains endospores green, and any other
bacterial bodies red.
• The green stain is malachite green,
• Counterstain is safranin, which dyes any other
bacterial bodies red
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
 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
 Success of Culture Methods

• Biology of organism (growth and oxygen

requirements)
• Site of infection
• Patient’s immune response
• Quality of culture media
• Appropriate collection and transport technique
 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. 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, Electrophoretic
Analysis of DNA
 5. Serologic Diagnosis
• Detect, identify and quantify antigen and
antibody response
• Titer
• Antigen test: Capsular swelling, Slide
agglutination test
• Antibody test: Immunoprecipitation
technoques, ELISA, Western blot, Compliment
fixation, latex agglutination, RIA
 Basic Issues in Proper Handling of
Specimens
• Collection of Specimens
• Important information includes:
* the specific site(s)
* whether the patient was receiving antibiotics prior 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.
 Basic Issues in Proper Handling of
Specimens
• 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 room temperature
* Neisseria species--transport rapidly to the
laboratory.