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Chapter 1

Bacterial Cell Structure,


Physiology, Metabolism, and
Genetics

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Case in Point
 A 4-year-old girl had the presenting
symptoms of redness, burning, and light
sensitivity in both eyes.
 Eyelids sticking together because of
exudative discharge
 Gram stain of the discharge showed gram-
positive intracellular and extracellular, faint-
staining, coccobacillary organisms. The
organisms appeared to have small, clear,
nonstaining “halos” surrounding each one.
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Significance
 Evolution
 Variation
 Role of clinical microbiologist

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Role of Clinical Microbiologist
 Culture organisms from specimens
 Snapshot of specimen
 Classification and identification of organisms
 Possible cause of disease
 Prediction and interpretation of susceptibility
 Improve treatment

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Bacterial Relationships to
Isolation
 Growth requirements of bacteria
 Allows microbiologist to select the correct medium
for primary culture
 Increases likelihood of pathogen isolation
 Steps in bacterial classification
 Determine staining characteristics
 Observe microscopic characterization of size and
shape
 Determine metabolic biochemical reactions

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Anton Von Leeuwenhoek
 Dutch lens maker and biologist
 Discovered “Beasties”
 “Father of Protozoology and Bacteriology”

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Overview of the Microbial World
 Bacteria
 Parasites
 Fungi
 Viruses

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Bacteria
 Prokaryotes
 No organelles
 Unicellular

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Prokaryotic Versus Eukaryotic
Organization

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Prokaryotic Versus Eukaryotic
Organization (Cont.)

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Prokaryotic Versus Eukaryotic
Organization (Cont.)

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Prokaryotic Versus Eukaryotic
Organization (Cont.)

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Illustration of Prokaryotic and
Eukaryotic Cells

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Parasites
 Eukaryotic
 Single or multicellular
 Motile or nonmotile
 Categorized by flagella, pseudopodia, or cilia

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Fungi
 Heterotrophic eukaryotes
 Yeast
 Unicellular, asexual reproduction
 Mold
 Most multicellular with sexual or asexual
reproduction
 Filamentous
 Dimorphic
 Filamentous at room temperature
 Yeast at human body temperature
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Viruses
 Smallest infectious particle
 Smaller than what is seen by light microscopes
 DNA or RNA
 May be single or double stranded
 Acellular
 Obligate intracellular parasites
 Require host cells for replication and are usually
host and/or host cell specific

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Viral Emergence
 Becoming better known by
 DNA or RNA makeup
 Host disease signs and symptoms
 Chemical makeup
 Geographic distribution
 Resistance to lipid solvents and detergents
 Resistance to pH and temperature changes
 Antigenicity (serologic methods)

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Viral Classification/Taxonomy
 Based on
 Genome
 Replication
 Virion structure

 International Committee on Taxonomy of


Viruses
 www.ncbi.nlm.nih.gov/ICTVdb/
 More than 2000

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Viral Classification/Taxonomy
(Cont.)
 Taxonomy is based on
 Genotype
• Base sequencing of DNA or RNA
• Comparison of the base composition ratio to determine
degree of relatedness
 Phenotype
• Macroscopic and microscopic morphology
• Staining characteristics
• Nutritional requirements
• Physiologic and biochemical characteristics
• Susceptibility or resistance to antibiotics or chemicals

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General Taxonomy
 Domain
 Kingdom
• Division (Phylum)
 Class
– Order

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General Taxonomy (Cont.)
 Order
 Family (-aceae)
• Tribe
 Genus (capitalized)
– species (lowercase) sp. singular or spp. plural

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Nomenclature
 Genus species or Genus species or G.
species
 Some genera have the same first letter so the
first syllable is used
 Staph. for staphylococcus
 Strept. for streptococcus
 Esch. coli (bacteria)
 Ent. coli (parasite)

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Classification by Phenotypic and
Genotypic Characteristics
 Species
 Subspecies (subsp.): phenotypic differences
• Serovarieties (serovar)
• Biovarieties (biovar)
• Phage typing: susceptibility to specific viruses
 Strain
 Species with different susceptibility patterns
• Example: susceptibility or resistance to penicillin

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Classification by Phenotypic and
Genotypic Characteristics (Cont.)
 Genetic relatedness
 rRNA
 Led to some reclassification

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Classification by Cellular Type:
Prokaryotes, Eukaryotes, and
Archaeobacteria
 Three domains
 Bacteria
• Classic prokaryotic cell encountered in clinical
microbiology
 Archaea
• Extremophiles
• Not encountered in clinical microbiology
 Eukarya
• More complex than prokaryotes
• Contain organelles

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Comparison of Cell Structure
 Eukaryotic cell structure cytoplasmic structure
 Cell envelope structures
• Plasma membrane
 Cell wall
 80S ribosomes (60S and 40S subunits)
 Prokaryotic cell structure
 Cell envelope structures
• Plasma membrane (cell membrane)

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Comparison of Cell Structure (Cont.)
 Prokaryotic cell structure
 Cell wall
• Gram-positive cell wall
• Acid-fast cell wall
• Gram-negative cell wall
• Absence of cell wall
 Surface polymers
 70S ribosomes (50S and 30S subunits)
• Svedberg (S) units are sedimentation rates during high
speed centrifugation.
• Values are NOT additive, due to binding together
resulting in surface area loss.
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Granules and Endospores
 Prokaryotes
 Granules in the cytoplasm
• Glycogen
• Poly-β-hydroxybutyrate
• Polyphosphates
 Endospores
• Bacillus and Clostridium produce endospores.
• Highly resistant to chemical agents, temperature change,
starvation, dehydration, ultraviolet (UV) light, gamma
radiation, and desiccation
• Not involved in reproduction

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Prokaryotes Versus Eukaryotes

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Prokaryotes Versus Eukaryotes
(Cont.)

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Prokaryotes Versus Eukaryotes
(Cont.)

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Prokaryotes Versus Eukaryotes
(Cont.)

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Prokaryotes Versus Eukaryotes
Visualized

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Prokaryotes Versus Eukaryotes
Visualized (Cont.)

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Functions of the Cell Membrane

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Gram Positive Versus Gram
Negative

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Gram Negative Outer Membrane
 Lipopolysaccharide components
 Antigenic O–specific polysaccharide
 Core polysaccharide
 Lipid A (also called endotoxin)

 Outer membrane functions


 Acts as a barrier to hydrophobic compounds and
harmful substances
 Acts as a sieve, allows water-soluble molecules to
enter through porins
 Enhances attachment to host cells

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Acid-Fast Cell Walls
 Waxy layer of glycolipids and fatty acids
 Major component is mycolic acid.
• Strongly hydrophobic
 Difficult to Gram stain: lightly gram-positive
 Acid-fast stain
 Carbolfuchsin
• Mycobacterium spp. and Nocardia spp. stain
• Others decolorize with acid-alcohol

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Bacteria Lacking Cell Walls
 Mycoplasma
 Ureaplasma
 Membrane contains sterols
 L-forms
 Bacteria that have lost cell walls

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Surface Polymers
 Capsule
 Organized polysaccharide or polypeptide structure
• Adherence to surface
• Prevents phagocytosis
 India ink
 Presents as clear halo-like structure
 Serologic typing
 Sometimes remove capsule to detect somatic
antigens, usually by boiling

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Cell Appendages
 Flagella
 External rotating filaments
 Lophotrichous
 Peritrichous

 Pili
 Nonmotile long filamentous tubes
 Fimbriae
 Hairlike proteins used for adhesion

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Cell Appendages (Cont.)

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Bacterial Morphology
 Bacteria size
 0.4 μm to 2 μm
 Microscopic shapes
 Cocci
 Bacilli
 Spiral

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Bacterial Morphology (Cont.)

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Common Stains
 Gram stain
 Acid-fast stains
 Acridine orange
 Methylene blue
 Lactophenol cotton blue
 Calcofluor white
 India ink
 Endospore stain

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Common Stains (Cont.)

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Gram Stain
 Heat fix (methanol can be used)
 Crystal violet (1 min)
 Primary stain
 Iodine (1 min)
 Fixes iodine
 Alcohol-acetone (quick on and rinse)
 Decolorizer
 Safranin (30 sec)
Counterstain
Note: Rinse with water between steps
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Acid-Fast Stain
 Ziehl-Neelsen method (heat)
 Kinyoun (detergent)
 Carbolfuchsin
• Primary stain (red)
 Acidified-alcohol
• Decolorizer
 Methylene blue
• Counterstain
 Auramine-rhodamine
 Fluorochrome stain
• Appears yellow or orange under fluorescent microscope
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Acridine Orange
 Stains nucleic acid orange under UV light
 Bacteria
 Living or dead
 Useful in samples with low bacterial numbers

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Calcofluor White
 Compound binds to chitin in fungi
 Bright apple-green and blue-white
fluorescence
 Requires UV light

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Other Stains
 Methylene blue
 Metachromatic granules in Corynebacterium
diphtheriae
 Lactophenol cotton blue
 Stains fungal cell walls blue
 India ink
 Negative stain to visualize capsules
 Endospore stain
 Malachite green stains endospores

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Microbial Growth and Nutrition
 Major nutritional needs
 Carbon source for cellular constituents
 Nitrogen source for proteins
 Adenosine triphosphate (ATP) energy source for
cell functions
 Trace elements
 Phosphorus (P), sulfur (S), sodium (Na),
potassium (K), chlorine (Cl), calcium (Ca)

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Microbial Growth and Nutrition
(Cont.)
 Microbial growth and nutrition
 Nutritional requirements for growth
• Autotroph
• Heterotroph
 Human pathogens

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Types of Growth Media
 Minimal medium
 Nutrient medium
 Enriched medium
 Selective medium
 Differential medium
 Transport medium

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Environmental Factors Influencing
Growth
 pH
 Human pathogens generally grow at neutral pH.
 Temperature
 Psychophiles
 Mesophiles
 Thermophiles

 Gaseous composition of the atmosphere


 Aerobes
• Obligate
• Facultative
 Anaerobes
• Obligate
• Aerotolerant
 Capnophiles
 Microaerophiles
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Bacterial Growth
 Generation time
 Time required for one cell to become two
 Growth curve
 Lag
 Log
 Stationary
 Death

 Determination of cell numbers


 Direct counts
 Plate counts
 Density
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Growth Curve

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Bacterial Biochemistry and
Metabolism
 Metabolism
 Fermentation and respiration
 Biochemical pathways from glucose to
pyruvic acid
 Anaerobic utilization of pyruvic acid
(fermentation)
 Aerobic utilization of pyruvate (oxidation)
 Carbohydrate utilization and lactose
fermentation

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Fermentation and Respiration
 Fermentation
 Anaerobic process carried out by both obligate and
facultative anaerobes
 Electron acceptor is an organic compound.
 Less efficient in energy generation than respiration
 Respiration
 Efficient energy-generating process in which
molecular oxygen is the final electron acceptor
 Certain anaerobes can carry out anaerobic
respiration.
• Inorganic forms of oxygen act as the final electron
acceptors.
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Three Major Pathways

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Three Major Pathways (Cont.)

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Three Major Pathways (Cont.)

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Embden-Meyerhof-Parnas (EMP)
Pathway

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Pentose Phosphate Pathway

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Entner-Doudoroff Pathway

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Anaerobic Utilization of Pyruvic
Acid (Fermentation)
 Alcoholic fermentation
 Homolactic fermentation
 Heterolactic fermentation
 Propionic fermentation
 Mixed acid fermentation
 Butanediol fermentation
 Butyric acid fermentation

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Bacterial Biochemistry and
Metabolism

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Aerobic Utilization of Pyruvate
(Oxidation)

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Carbohydrate Utilization and
Lactose Fermentation
 Enterobacteriaceae family
 Lactose differentiates organisms
• β-galactoside permease
• β-galactosidase
 Breaks bonds of glucose to galactose
– Releases glucose

 Other organisms

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Bacterial Genetics
 Anatomy of a DNA and RNA molecule
 Double helix
 Phosphate-pentose sugar-nitrogen containing
base
• Deoxyribose or ribose
• Purine (A or G) or pyrimidine (T or U and C)
 A=T C=G in DNA
 A=U C=G in RNA

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Anatomy of a DNA and RNA
Molecule

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Replication

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Terminology
 DNA
 Storage of genetic information (genetic potential)
 Replication produces DNA copies.

 RNA
 Produced by transcription of DNA
 mRNA

 Translation
 mRNA is read by ribosome.
• Codon: a group of three nucleotides
• tRNA matches codon with anticodon
 Protein
 Functional unit: expression of genetic potential
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Bacterial Genetics
 Genetic elements and alterations
 The bacterial genome
 Extrachromosomal elements
• Plasmids
 Mobile genetic elements
• Mutations
• Genetic recombination

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Bacterial Genetics (Cont.)
 Mechanisms of gene transfer
 Transformation
• Uptake and incorporation of naked DNA
 Transduction
• Transfer of genes by a bacteriophage
 Conjugation
• Transfer of genetic material from a donor to a recipient
strain of bacteria
 Restriction enzymes
• Enzymes that cut DNA at specific sequences

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Mechanisms of Gene Transfer

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