THE SCIENCE OF

MICROBIOLOGY
MARGIE S. GAYAPA, M.D., DPSP
SECTION OF MICROBIOLOGY AND PARASITOLOGY
COLLEGE OF MEDICINE
CENTRAL PHILIPPINE UNIVERSITY
MICROBIOLOGY
• Study of microorganisms

• Microbes, or microorganisms are minute

living things that are usually unable to be
viewed with the naked eye
MICROBES - What comes to mind?

• Diseases
• Infections
• Epidemics
• Food Spoilage

• Only 1% of all known bacteria cause human diseases

• 95% of known bacteria are non-pathogenic
 BENEFIT TO HUMANS

1. Bacteria are primary decomposers - recycle nutrients back into the

environment (sewage treatment plants)

2. Microbes produce various food products

• cheese, pickles, sauerkraut, green olives
• yogurt, soy sauce, vinegar, bread
• beer, wine, alcohol
BENEFIT TO HUMANS

3. Microbes are used to produce antibiotics

4. Bacteria synthesize chemicals that our body needs

but cannot synthesize
• Vitamin B
• Vitamin K
 BENEFIT TO HUMANS

5. Recombinant DNA Technology

Gene Therapy
Genetic Engineering

Bacteria can be manipulated to produce enzymes and proteins they

normally would not produce
• Insulin
• Human Growth Hormone
• Interferon
 MICROBIOLOGY

Bacteria Bacteriology
Protozoans Protozoology
Algae Phycology
Fungi and Slime Molds Mycology
Viruses Virology
Prions
 PRIONS
(Proteinaceous Infectious Particles)

PrPc PrPres
Sensitive soluble detergent
Proteases (transmissibility)

Dse causing conformation

 Prion Diseases
• Transmissible spongiform encephalopathies (TSEs)

• Rare progressive neurodegenerative disorders that

affect both humans and animals

• Distinguished by long incubation periods,

characteristic spongiform changes associated with
neuronal loss, and a failure to induce inflammatory
response

Long incubation
Neuronal loss
Do not iniate inflammatory
response
VIRUSES

- vary in diameter
from 15 to 300 nm

- consist of: nucleic

acid core, capsid/
protein coat,
envelope
 VIRUSES

• Differ from other microbes in at least two ways:

• Simple and acellular (obligate intracellular parasites)
• Absence of both DNA and RNA in the same virion

• Differ viriods
Only RNA
NO PROTEIN COAT
Property Prokaryotes Eukaryotes
Organization of Genetic Material
True membrane-bound nucleus Absent Present
DNA complexed with histones No Yes
Number of chromosomes One >1
Nucleolus Absent Present
Mitosis occurs No Yes
Genetic Recombination Partial, unidirectional transfer of Meiosis and fusion of gametes
DNA
Mitochondria Absent Present
Chloroplasts Absent Present
Plasma Membrane with Sterols Usually no Yes
Flagella Submicroscopic in size, Microscopic, membrane-
composed of one fiber bound, usually 20
microtubules in 9+2

Endoplasmic Reticulum Absent Present

Golgi Apparatus Absent Present
Cell Walls Chemically Chemically simpler and lacking
complex with peptidoglycan peptidoglycan

Differences in Simpler Organelles 50&30S 60S&40S

Ribosomes 70s 80s
Lysosomes and peroxisomes Absent Present
Microtubules Absent or rare Present
Differentiation Rudimentary Tissues and Organs
 PROKARYOTES

• Clone: Population of cells derived from a single cell

• Quorum Sensing: Cell to cell communication mechanism to regulate
transcription of genes
• Plasmids: Small, specialized genetic elements capable of replication
• Bacterial symbionts/ endosymbionts: Chloroplasts and mitochondria

Colony size
CLASSIFICATION OF PROKARYOTES
• Structural
• Physiologic
• Biochemical
• Genetic: Recombinant DNA technology (DNA probes) –
phylogenetic relationships
• Major Subdivisions: Bacteria and Archaebacteria
Classification of Microbes
 PROTISTS
(Microbial Eukaryotes)
• Algae – photosynthetic, many are unicellular;
dinoflagellates, Gonyaulax species

• Protozoans – unicellular, non-photosynthetic

• Fungi - non-photosynthetic, grow as a mass of hyphae

(mycelium)

• Slime molds – characterized by the presence of an

amoeboid multinucleate mass of cytoplasm called
plasmodium
 CLASSIFICATION
OF BACTERIA

MARGIE S. GAYAPA, M.D., DPSP

SECTION OF MICROBIOLOGY AND PARASITOLOGY
WEST VISAYAS STATE UNIVERSITY
CLASSIFICATION
OF BACTERIA
• Taxonomy: classification of organisms in
an ordered system that indicates a natural
relationship

• Classification: categorization of
organisms into taxonomic groups
• Nomenclature: naming of organism by
international rules
• Identification: practical use of a
classification scheme to isolate and
distinguish organisms/ causative agent
Five Kingdoms
Uni or multicellular eukaryotic

Eukaryotic
unicellular
Bacteria Prokaryotic
Archaea unicellular
23
CLASSIFICATION OF BACTERIA
Taxonomic Hierarchy/ Ranks
Domain
Kingdom Binomial
nomenclature uses
Phylum the Genus and
Class Species name to
Order identify each
creature.
Family
Genus
Species
CRITERIA FOR CLASSIFICATION
OF BACTERIA

• Growth on Media
• Bacterial Microscopy
• Biochemical Tests
• Immunologic Tests
• Genetic Instability
Based on Growth on Media
• Nonselective media
• BAP, chocolate agar

• Selective media
• Mac Conkey, CNA
• Inhibits the growth of some
bacteria while selecting for
the growth of others

• Differential media
• BAP, Mac Conkey
Based on Growth on Media
Based on Growth on Media
Based on Bacterial Microscopy
Four Groups Based on Cell
Wall Composition

1. Gram-positive

2. Gram-negative

3. Bacteria without
cell walls

4. Bacteria with
chemically unique
cell walls
 Based on Biochemical Tests
OXIDASE TEST
• Distinguish
organisms based on
the presence of the
respiratory enzyme
cytochrome C
• Differentiates
Enterobacteriaceae
from other gram-
negative rods
CATALASE TEST
 Based on Immunologic Tests

• Subdivide strains of a particular

bacterial species
 Based on Genetic Criteria
• Investigate relatedness of genes by
comparing sequences among different
bacteria

• GENETIC INSTABILITY - cause some

traits to be highly variable within
biologic groups or within a specific
taxonomic group

• Carried by plasmids or bacteriophages

• Antibiotic resistance genes
• Genes carrying enzymes
CLASSIFICATION
SYSTEMS
PHYLOGENETIC CLASSIFICATION
• Measures between two organisms and imply
that they share a common ancestor

• Species: genomically coherent group of

individual isolates or strains sharing a high
degree of similarity in many independent
features under highly standardized
conditions
• Chemical characterization based on the
nucleotide base composition - (G+C) content
• DNA sequencing: more precise
rRNA Sequence
• The greatest advancement in classifying organisms
• Differences in the nucleotide sequence are used to
classify prokaryotes
• 16S rRNA sequences
• 23S rRNA sequences
Actually look at the DNA that
codes for the rRNA
3CarlDomain
Woese
System
Late 1970’s & early 1980’s
Woese, C. R. 2004. A New Biology for a New Century. MMBR. June 68(2):173-86.
Woese, C.R. 2002. On the evolution of cells. Proc Natl Acad Sci USA 99(13):8742-7.
Woese, C.R. 2000. Interpreting the universal phylogenetic tree. Proc Natl Acad Sci USA 97(15):839
39
Numerical Taxonomy
Looked at all traits of an organism - every
trait had equal importance (Analytical
Profile Index)
Bergey’s Manual of Systematic
Bacteriology
• Prokaryotes into 25 phyla
• Archaeabacteria (2)
• Eubacteria (23)

• Consensus of experts
 SUBTYPING
• Important to identify strains of a given
species

• Serological Typing: monoclonal

antibodies against cell surface antigens
to identify bacterial serotypes/
serovars; genotyping multi-locus
enzyme electrophoresis (MLEE)

• Chemical Fingerprinting: FTIR,

pyrolysis, mass spectrometry, Maldi-tof
Fourier transform infrared spectroscopy
(FTIR),

matrix-assisted laser
desorption/ionization with time-of-flight
NUCLEIC ACID-BASED TAXONOMY
• Plasmid Analysis: most useful for
examining outbreaks that are restricted
to time and place; separated by
electrophoresis
• Restriction Endonucleases Analysis
• Southern Blotting
• Ribotyping: uses Southern Blot
analysis to detect polymorphisms of
rRNA gene
• Repetitive Sequences: useful in
subtyping B. antracis, Y. pestis,
F. tularensis
 Southern Blotting
RIBOTYPING

DNA preparations from bacterial isolates

are subjected to restriction endonuclease method uses
digestion. After agarose gel Southern blot
electrophoresis, the separated restriction analysis to
fragments are transferred to a detect
nitrocellulose or nylon membrane. These polymorphism
double-stranded DNA fragments are first s of rRNA
converted into single-stranded linear genes
sequences. Using a labeled fragment of
DNA as a probe, it is possible to identify
the restriction fragments containing
sequences (loci) that are homologous to
the probe by complementation to the
bound single-stranded fragments
 CELL
STRUCTURE
MARGIE S. GAYAPA, M.D., DPSP
SECTION OF MICROBIOLOGY AND PARASITOLOGY
WEST VISAYAS STATE UNIVERSITY
Bright-field Microscope
Phase-Contrast
Microscope
Dark-Field Microscope
Fluorescence Microscope
Electron Microscope (TEM)
Electron Microscope
EUKARYOTES
• Nucleus – contains the genome
• DNA is linear and complexed with histones
• Nucleolus – rich in rRNA
• Nuclear membrane – exhibits selective
permeability

• Organelles
• RER – major producer of glycoprotein
• SER – lipid synthesis, CHO metabolism
EUKARYOTES
• Organelles
• Golgi apparatus – modify and sort products
of ER
• Plastids (mitochondria and chloroplasts) –
contains 70s ribosomes, DNA
• Lysosomes - digest macromolecules for
nutrients
• Peroxisomes – produce hydrogen peroxide
EUKARYOTES
• Cytoskeleton – 3 dimensional structure that fills
the cytoplasm structure:
• Microfilaments – subunits of actin, defines
and maintain shape; gliding, contraction and
cytokinesis
• Intermediate filaments – provides tensile
strength
• Microtubules – tubulin, assist microfilaments
in maintaining the structure

• Cilia and Flagella – tubulin, “9+2” arrangement

 External Structures
• Appendages
• Two major groups of appendages:
• Motility – flagella and axial filaments
(periplasmic flagella)
• Attachment or channels – fimbriae and pili
• Glycocalyx – surface coating
 Flagella
• 3 parts:
• filament – long, thin, helical structure composed of
protein flagellin
• hook- curved sheath
• basal body – stack of rings firmly anchored in cell
wall
• Rotates 360o
• Number and arrangement of flagella varies:
• monotrichous, lophotrichous, amphitrichous,
peritrichous
• Functions in motility of cell through
environment
 Flagellar Arrangements
1. Monotrichous – single flagellum at one end
2. Lophotrichous – small bunches arising from one
end of cell
3. Amphitrichous – flagella at both ends of cell
4. Peritrichous – flagella dispersed over surface of
cell; slowest
Flagellar Function
Guide bacteria in a direction in response to
external stimulus:
chemical stimuli – chemotaxis; positive and
negative
light stimuli – phototaxis
Signal sets flagella into rotary motion clockwise
or counterclockwise:
counterclockwise – results in smooth linear
direction – run
clockwise - tumbles
 Axial Filaments
• Periplasmic, internal
flagella, enclosed between
cell wall and cell
membrane of spirochetes

• Produce cellular motility

by contracting and
imparting twisting or
flexing motion
 Pili
• Rigid tubular structure made of pilin protein
• Found only in Gram-negative bacteria
• Ordinary pilus
• Sex pilus - Function to join bacterial cells for partial DNA
transfer called conjugation
 Glycocalyx
• Coating of molecules external to the cell
wall, made of sugars and/or proteins
• Two types:
1. slime layer - loosely organized and
attached
2. capsule - highly organized, tightly
attached
• Functions:
• protect cells from dehydration and nutrient
loss
• inhibit killing by white blood cells by
phagocytosis contributing to pathogenicity
• attachment - formation of biofilms
 The Cell Envelope
• External covering outside the cytoplasm
• Composed of two basic layers:
• cell wall and cell membrane
• Maintains cell integrity
• Two generally different groups of bacteria
demonstrated by Gram stain:
• Gram-positive bacteria: thick cell wall composed
primarily of peptidoglycan and cell membrane
• Gram-negative bacteria: outer cell membrane, thin
peptidoglycan layer, and cell membrane
Insert figure 4.12
Comparative cell envelopes
 Structure of Cell Walls
• Determines cell shape, prevents lysis (bursting)
or collapsing due to changing osmotic pressures
• Peptidoglycan is primary component:
• unique macromolecule composed of a
repeating framework of long glycan chains
cross-linked by short peptide fragments
 Gram-positive Cell Wall
• Thick, homogeneous sheath of peptidoglycan
• 20-80 nm thick
• includes teichoic acid (capsular polymers
containing glycerophosphates and ribitol
phosphates residues) and teichuronic acids
(sugar acids as repeat units) --- function in cell
wall maintenance and enlargement during cell
division; move cations across the cell envelope;
stimulate a specific immune response

• Wall teichoic acid

• Membrane teichoic acid/ LTA
 Gram-negative Cell Wall

• Single, thin sheet of peptidoglycan

• Protective structure while providing some
flexibility and sensitivity to lysis
 Gram-negative Cell Envelope
• Composed of an outer membrane, a thin
peptidoglycan layer, and a cell membrane
• Outer membrane is similar to cell membrane
bilayer structure
• outermost layer contains lipopolysaccharides and
lipoproteins (LPS)
• Lipid A: B-OH myristic acid
• Core polysaccraride: KDO, hexose
• O antigen
Ketodeoxyoctanic acid
 Atypical Cell Walls
• Some bacterial groups lack typical cell wall structure
i.e. Mycobacterium and Nocardia
• Gram-positive cell wall structure with lipid mycolic
acid (cord factor)
• pathogenicity and high degree of resistance to
certain chemicals and dyes
• basis for acid-fast stain used for diagnosis of
infections caused by these microorganisms
• Some have no cell wall i.e. Mycoplasma
• cell membrane is stabilized by sterols
• pleomorphic
• versus protoplasts, spheroplasts and L forms
 Cell Membrane Structure
• Phospholipid bilayer with embedded proteins – fluid
mosaic model
• Functions in:
• Selective permeability and transfer of solutes
• Electron transport and oxygen phosphorylation
• Excretion of hydrolytic enzymes
• Bearing the enzyme and carrier molecules that
function in the synthesis of DNA, cell wall
polymers and membrane lipids
• Bearing the receptors and other proteins of the
chemotactic and other sensory transduction
proteins
Membrane Transport
• Passive transport
• Simple diffusion
• Facilitated diffusion – selective, involves
channel proteins that facilitate passage of
specific molecules
• Active transport
• Ion coupled transport – previously established
ion gradient e.g. proton-motive or sodium-
motive force; three types: uniport, symport,
antiport
• ATP binding cassette (ABC) – hydrolysis of
ATP, the energy is used to open the pore and
allow unidirectional movement of substrate
Membrane Transport
• Group translocation – coupling transport with
metabolism e.g. phosphotransferase system
• Special transport processes – e.g, siderophores
 Bacterial Internal Structures
• Cell cytoplasm:
• dense gelatinous solution of sugars, amino acids,
and salts
• 70-80% water
• serves as solvent for materials used in all cell
functions
 Bacterial Internal Structures
• Chromosome
• single, circular, double-stranded DNA
molecule that contains all the genetic
information required by a cell
• DNA is tightly coiled around a protein,
aggregated in a dense area called the nucleoid
 Bacterial Internal Structures
• Plasmids
• small circular, double-stranded DNA
• free or integrated into the chromosome
• duplicated and passed on to offspring
• not essential to bacterial growth and metabolism
• may encode antibiotic resistance, tolerance to
toxic metals, enzymes and toxins
• used in genetic engineering- readily manipulated
and transferred from cell to cell
Bacterial Internal Structures
• Ribosomes
• made of 60% ribosomal RNA and 40% protein
• consist of two subunits: large and small
• procaryotic differ from eucaryotic ribosomes
in size and number of proteins
• site of protein synthesis
• present in all cells
 Bacterial Internal Structures
• Inclusions and granules
• intracellular storage bodies
• vary in size, number and content
• Bacterial cell can use them when environmental
sources are depleted.
• examples: glycogen, poly-b-hydroxybutyrate, gas
vesicles for floating, sulfur and phosphate
granules (metachromatic granules)
 Bacterial Internal Structures
• Endospores
• inert, resting, cells produced by some G+ genera:
Clostridium, Bacillus and Sporosarcina
• have a 2-phase life cycle:
• vegetative cell – metabolically active and growing
• endospore – when exposed to adverse
environmental conditions; capable of high
resistance and very long-term survival
• sporulation -formation of endospores
• hardiest of all life forms
• withstands extremes in heat, drying, freezing,
radiation and chemicals
• not a means of reproduction
• germination- return to vegetative growth
Endospores
• Resistance linked to high levels of calcium
and dipicolinic acid
• Dehydrated, metabolically inactive
• Thick coat
• Longevity verges on immortality - 25,250
million years.
• Resistant to ordinary cleaning methods
and boiling
• Pressurized steam at 120oC for 20-30
minutes will destroy

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