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微生物學. 許勝傑 博士 長庚大學 生物醫學系 助理教授 E-mail: 電話 (03)211-8800#3690. Chapter 1 The Evolution of Microorganisms and Microbiology. CHAPTER GLOSSARY Archaea Bacteria Eukarya Fungi Genome Genomic analysis Koch’s postulates Microbiology Microorganism Prions

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許勝傑 博士


生物醫學系 助理教授


電話 (03)211-8800#3690


Chapter 1

The Evolution of Microorganisms and Microbiology







Genomic analysis

Koch’s postulates




Prokaryotic cells


Spontaneous generation




Figure/ Table



What is microbiology?

  • study of organisms too small to be clearly seen by the unaided eye (i.e., microorganisms)
  • these organisms are relatively simple in their construction and lack highly differentiated cells and distinct tissues

The Importance of Microorganisms

  • most populous and diverse group of organisms
  • found everywhere on the planet
  • play a major role in recycling essential elements
  • source of nutrients and some carry out photosynthesis
  • benefit society by their production of food, beverages, antibiotics, and vitamins
  • some cause disease in plants and animals

Microbes are estimated to contains 50% of biological carbon and 90% of biological nitrogen on Earth.


Fig. 1.1 Concept map showing the types of biological entities studied by microbiologists.

Members of the microbial world
  • prokaryoticcells lack a true membrane-delimited nucleus
  • eukaryoticcells have a membrane-enclosed nucleus, are more complex morphologically and are usually larger than prokaryotic cells

Classification schemes

  • five kingdom scheme includes Monera原核界, Protista, Fungi, Animalia and Plantae with microbes placed in the first three kingdoms
  • three domain alternative, based on a comparison of ribosomal RNA, divides microorganisms into Bacteria(true bacteria),Archaeaand Eucarya (eucaryotes)
discovery of microorganisms
Discovery of Microorganisms
  • Antony van Leeuwenhoek (1632-1723)
    • first person to observe and describe microorganisms accurately
the conflict over spontaneous generation
The Conflict over Spontaneous Generation
  • spontaneous generation
    • living organisms can develop from nonliving or decomposing matter
  • Francesco Redi (1626-1697)
    • disproved spontaneous generation for large animals
    • showed that maggots on decaying meat came from fly eggs
louis pasteur 1822 1895
Louis Pasteur (1822-1895)
  • his experiments
    • placed nutrient solution in flasks
    • created flasks with long, curved necks
    • boiled the solutions
    • left flasks exposed to air
  • results: no growth of microorganisms
koch s postulates
Koch’s postulates
  • The microorganism must be present in every case of the disease but absent from healthy individuals.
  • The suspected microorganism must be isolated and grown in a pure culture.
  • The same disease must result when the isolated microorganism is inoculated into a healthy host.
  • The same microorganism must be isolated again from the diseased host.

Earliest Molecules - RNA

  • original molecule must have fulfilled protein and hereditary function
  • ribozymes
    • RNA molecules that form peptide bonds
    • perform cellular work and replication
  • earliest cells may have been RNA surrounded by liposomes

Chapter 2

The Study of Microbial Structure: Microscopy and Specimen Preparation


Atomic force microscope

Bright-field microscope

Confocal scanning laser microscope (CSLM)

Dark-field microscope

Differential interference contrast (DIC) microscopy

Differential staining


Fluorescence microscopy

Gram stain

Negative staining


Phase-contrast microscope

Refractive index


Scanning electron microscope (SEM)

Simple staining

Transmission electron microscope (TEM)

  • focus light rays at a specific place called the focal point
  • distance between center of lens and focal point is the focal length
  • strength of lens related to focal length
    • short focal length more magnification
the light microscope
The Light Microscope
  • bright-field microscope
  • dark-field microscope
  • phase-contrast microscope
  • fluorescence microscope
microscope resolution
Microscope Resolution
  • ability of a lens to separate or distinguish small objects that are close together
  • wavelength of light used is major factor in resolution

shorter wavelength  greater resolution

preparation and staining of specimens


  • preserves internal and external structures and fixes them in position
  • organisms usually killed and firmly attached to microscope slide
    • heat fixation – routine use with procaryotes
      • preserves overall morphology but not internal structures
    • chemical fixation – used with larger, more delicate organisms
      • protects fine cellular substructure and morphology
Preparation and Staining of Specimens
  • increases visibility of specimen
  • accentuates specific morphological features
  • preserves specimens
dyes and simple staining
Dyes and Simple Staining
  • dyes
    • Ionizable dyes have charged groups
      • basic dyes have positive charges
      • acid dyes have negative charges
      • chromophore groups
        • chemical groups with conjugated double bonds
  • simple stains
    • a single stain is used
    • use can determine size, shape and arrangement of bacteria
gram staining
Gram staining

Differential Staining

  • divides microorganisms into groups based on their staining properties
  • divides bacteria into two groups based on differences in cell wall structure
electron microscopy
Electron Microscopy
  • beams of electrons are used to produce images
  • wavelength of electron beam is much shorter than light, resulting in much higher resolution
newer techniques in microscopy
Newer Techniques in Microscopy
  • confocal scanning laser (CLSM) microscopy and scanning probe microscopy
  • have extremely high resolution
confocal microscopy
Confocal Microscopy
  • laser beam used to illuminate a variety of planes in the specimen
  • computer compiles images created from each point to generate a 3-dimensional image
  • used extensively to observe biofilms
scanning probe microscopy
Scanning Probe Microscopy
  • atomic force microscope
    • sharp probe moves over surface of specimen at constant distance
    • up and down movement of probe as it maintains constant distance is detected and used to create image

Chapter 3

Bacteria and Archaea


Lipopolysaccharides (LPSs)



Periplasmic space


Porin proteins

Sex pili






Cell envelope





Fluid mosaic model

Gas vacuole




Common features of bacterial and archaeal cell structure

  • prokaryotes differ from eukaryotes in size and simplicity
    • most lack internal membrane systems
  • prokaryotes are divided into Bacteria and Archaea
  • Bacteria are divided into 2 groups based on their Gram stain reaction
size shape and arrangement
Size, Shape, and Arrangement
  • cocci (s., coccus) – spheres
    • diplococci (s., diplococcus) – pairs
    • streptococci – chains
    • staphylococci – grape-like clusters
    • tetrads – 4 cocci in a square
    • sarcinae – cubic configuration of 8 cocci
  • bacilli (s., bacillus) – rods
    • coccobacilli – very short rods
    • vibrios – resemble rods, comma shaped
  • spirilla (s., spirillum) – rigid helices
  • Spirochetes 螺旋體– flexible helices
  • Mycelium 菌絲– network of long, multinucleate filaments
  • Pleomorphic多形性– organisms that are variable in shape
  • Archaea
    • pleomorphic, branched, flat, square, other unique shapes

largest –

  • 50 μm in
  • diameter
  • smallest –
  • 0.3 μm in
  • diameter

Size – Shape Relationship

  • important for nutrient uptake
  • surface to volume ratio (S/V)
  • small size may be protective mechanism from predation掠食
bacterial cell envelope
Bacterial Cell Envelope
  • Plasma membrane
  • Cell wall
  • Layers outside the cell wall

Functions of the plasma membrane

  • encompasses the cytoplasm
  • selectively permeable barrier
  • interacts with external environment
    • receptors for detection of and response to chemicals in surroundings
    • transport systems
    • metabolic processes
fluid mosaic model of membrane structure
Fluid Mosaic Model of Membrane Structure
  • Lipid bilayerin which proteins float

Membrane proteins

  • peripheral proteins
    • loosely associated with the membrane and easily removed
  • integral proteins
    • embedded within the membrane and not easily removed
the asymmetry of most membrane lipids
The asymmetry of most membrane lipids
  • polar ends
    • interact with water
    • hydrophilic
  • nonpolar ends
    • insoluble in water
    • hydrophobic

Bacterial Membranes

  • differ from eukaryotes in lacking sterols
    • do contain hopanoids, sterol-like molecules
  • a highly organized, asymmetric system which is also flexible and dynamic
the bacteria cell wall
The Bacteria Cell Wall
  • rigid structure that lies just outside the plasma membrane

Functions of cell wall

  • provides characteristic shape to cell
  • protects the cell from osmotic lysis
  • may also contribute to pathogenicity
  • very few procaryotes lack cell walls
bacterial cell walls
Bacterial Cell walls
  • bacteria are divided into two major groups based on the response to gram-stain procedure.
    • gram-positive bacteria stain purple
    • gram-negative bacteria stain pink
  • staining reaction due to cell wall structure
peptidoglycan murein structure
Peptidoglycan (肽聚糖)(Murein, 胞壁質) Structure
  • meshlike polymer of identical subunits forming long strands
    • two alternating sugars
      • N-acetylglucosamine (NAG) (N-乙酰葡萄糖胺)
      • N- acetylmuramic acid (NAM) (N-乙酰胞壁酸)
    • alternating D- and L- amino acids

Diaminoacids present in peptidoglycan


peptidoglycan strands have a helical shape

  • peptidoglycan chains are crosslinked by peptides for strength



gram positive cell walls
Gram-Positive Cell Walls
  • composed primarily of peptidoglycan
  • may also contain large amounts of teichoic acids (negatively charged)
    • help maintain cell envelop
    • protect from environmental substances
    • may bind to host cells
  • some gram-positive bacteria have layer of proteins on surface of peptidoglycan
  • teichoic acids
  • polymers of glycerol or ribitol (核糖醇) joined by phosphate groups

Isolated gram+ cell wall

periplasmic space of gram bacteria
Periplasmic Space of Gram + bacteria
  • lies between plasma membrane and cell wall and is smaller than that of Gram - bacteria
  • periplasm has relatively few proteins
  • enzymes secreted by Gram + bacteria are called exoenzymes
    • aid in degradation of large nutrients
gram negative cell walls
Gram-Negative Cell Walls
  • consist of a thin layer of peptidoglycan surrounded by an outer membrane
  • outer membrane composed of lipids, lipoproteins, and lipopolysaccharide (LPS)
  • no teichoic acids
  • peptidoglycan is ~5-10% of wall weight
  • periplasmic space differs from that in Gram + cells
    • may constitute 20-40% of cell volume
lipopolysaccharides lpss
Lipopolysaccharides (LPSs)
  • consists of three parts
    • lipid A
    • core polysaccharide
    • O side chain (O antigen)

Importance of LPS

  • may contribute to attachmentto surfaces and biofilm formation
  • creates apermeability barrier
  • contributes to negative charge on cell surface (core polysaccharide)
  • protection from host defenses (O antigen)
  • helps stabilizeouter membrane structure (lipid A)
  • can act as an endotoxin(lipid A)
other characteristics of the outer membrane
Other Characteristics of the Outer Membrane
  • more permeable than plasma membrane due to presence of porin proteins and transporter proteins
    • porin proteins form channels through which small molecules (600-700 daltons) can pass
the mechanism of gram staining
The Mechanism of Gram Staining
  • thought to involve shrinkage of the pores of the peptidoglycan layer of gram-positive cells
    • constriction prevents loss of crystal violet during decolorization step
  • thinner peptidoglycan layer and larger pores of gram-negative bacteria does not prevent loss of crystal violet

The Cell Wall and Osmotic Protection

  • osmotic lysis
    • can occur when cells are in hypotonicsolutions
    • movement of water into cell causes swelling and lysis due to osmotic pressure
  • cell wall protects against osmotic lysis
  • lysozymebreaks the bond between N-acetyl glucosamine and N-acetylmuramic acid
  • penicillin inhibits peptidoglycan synthesis
  • If cells are treated with either of the above they will lyse if they are in a hypotonic solution
  • protoplast – cell completely lacking cell wall
  • spheroplast – cell with some cell wall remaining
capsules slime layers and s layers
Capsules, Slime Layers, and S-Layers
  • capsules
    • usually composed of polysaccharides
    • well organized and not easily removed from cell
  • slime layers (黏液層)
    • similar to capsules except diffuse, unorganized and easily removed
    • slime may aid in motility
  • S-layers
    • regularly structured layers of protein or glycoprotein
    • In bacteria the S layer is external to the cell wall
    • common among Archaea, where they may be the only structure outside the plasma membrane


functions of capsules slime layers and s layers
Functions of capsules, slime layers, and S-layers
  • protection from host defenses (e.g., phagocytosis)
  • protection from harsh environmental conditions (e.g., desiccation)
  • attachment to surfaces

More functions…

  • protection from viral infection or predation by bacteria
  • protection from chemicals in environment (e.g., detergents)
  • facilitate motility of gliding bacteria
  • protection against osmotic stress

Glycocalyx (醣外被)

    • network of polysaccharides extending from the surface of the cell
    • a capsule or slime layer composed of polysaccharides can also be referred to as a glycocalyx
archaeal membranes
Archaeal membranes
  • composed of unique lipids
    • isoprene units (five carbon, branched)
    • ether linkages rather than ester linkages to glycerol
  • some have a monolayer structure instead of a bilayer structure
archaeal cell walls differ from bacterial cell walls
Archaeal Cell Walls Differ from Bacterial Cell Walls
  • lack peptidoglycan
  • most common cell wall is S layer
  • may have protein sheath external to S layer
  • S layer may be outside membrane and separated by pseudomurein
  • pseudomurein may be outermost layer – similar to gram-positive microorganisms
archaeal cell walls
Archaeal cell walls
  • lack peptidoglycan
  • cell wall varies from species to species but usually consists of complex heteropolysaccharides
  • Methanogens have walls containing pseudomurein


Cell envelopes of Archaea


Bacterial and ArchaealCytoplasmic Structures

  • Cytoskeleton
  • Intracytoplasmic membranes
  • Inclusions
  • Ribosomes
  • Nucleoid and plasmids
cytoplasmic matrix
Cytoplasmic Matrix
  • substance in which nucleoid, ribosomesand inclusion bodies are suspended
  • lacks organelles bound by unit membranes
  • composed largely of water
  • is a major part of the protoplasm (the plasma membrane and everything within)

The Procaryotic Cytoskeleton

  • homologs of all 3 eucaryotic cytoskeletal elements have recently been identified in Bacteria and one has been found in Archaea
  • functions include roles in cell division, protein localization and determination of cell shape


  • FtsZ – many bacteria and archaea
    • forms ring during septum formation in cell division
  • MreB – many rods, some archaea
    • maintains shape by positioning peptidoglycan synthesis machinery
  • CreS – rare, maintains curve shape


Intermediate filament

intracytoplasmic membranes
Intracytoplasmic Membranes
  • plasma membrane infoldings
    • observed in many photosynthetic bacteria
      • analogous to thylakoids of chloroplasts
      • reactions centers for ATP formation
    • observed in many bacteria with high respiratory activity
  • anammoxosome in Planctomycetes
    • organelle – site of anaerobic ammonia oxidation
  • granules of organic or inorganic material that are stockpiled by the cell for future use
  • some are enclosed by a single-layered membrane
    • membranes vary in composition
    • some made of proteins; others contain lipids

Organic inclusion bodies

  • glycogen
    • polymer of glucose units
  • poly-β-hydroxybutyrate (PHB)
    • polymers of β-hydroxybutyrate
  • cyanophycin granules
    • large polypeptides containing about equal quantities of arginine and aspartic acid
  • carboxysomes
    • contain the enzyme ribulose-1,5,-bisphosphate carboxylase (Rubisco), enzyme used for CO2 fixation
gas vacuoles
    • found in cyanobacteria and some other aquatic procaryotes
    • provide buoyancy
    • aggregates of hollow cylindrical structures called gas vesicles
inorganic inclusion bodies
Inorganic inclusion bodies
  • polyphosphate granules
    • also called volutin granules and metachromatic granules
    • linear polymers of phosphates
  • sulfur granules
  • Magnetosomes Fe3O4
    • found in aquatic bacteria
    • magnetite particles for orientation in Earth’s magnetic field
    • cytoskeletal protein MamK
    • helps form magnetosome chain
  • complex structures consisting of protein and RNA
  • sites of protein synthesis
  • smaller than eucaryoticribosomes
    • procaryoticribosomes 70S
    • eucaryoticribosomes  80S
      • S = Svedburg unit

bacterial and archaeal ribosomal RNA

    • 16S small subunit
    • 23S and 5S in large subunit
    • archaea has additional 5.8S (also seen in eukaryotic large subunit)
  • proteins vary
    • archaea more similar to eukarya than to bacteria
the nucleoid
The Nucleoid (類核體)

Procaryotic chromosomes are located in the nucleoid, an area in the cytoplasm

  • irregularly shaped region
  • location of chromosome
    • usually 1/cell
  • not membrane-bound
the procaryotic chromosome
The procaryotic chromosome
  • a closed circular, double-stranded DNA molecule
  • looped and coiled extensively
  • nucleoid proteins probably aid in folding
    • nucleoid proteins differ from histones


  • usually small, closed circular DNA molecules
  • exist and replicate independently of chromosome
  • have relatively few genes present
  • genes on plasmids are not essential to host but may confer selective advantage (e.g., drug resistance)
  • curing is the loss of a plasmid
  • classification of plasmids based on mode of existence, spread, and function




External Structures


protection, attachment to surfaces, horizontal gene transfer, cell movement

pili and fimbriae
Pili and Fimbriae
  • fimbriae (s., fimbria)
    • short, thin, hairlike, proteinaceous appendages
      • up to 1,000/cell
    • mediate attachment to surfaces
    • some (type IV fimbriae) required for twitching motility or gliding motility that occurs in some bacteria
  • sex pili (s., pilus)
    • similar to fimbriae except longer, thicker, and less numerous (1-10/cell)
    • required for mating
patterns of flagella distribution
Patterns of Flagella Distribution
  • monotrichous – one flagellum
  • polar flagellum – flagellum at end of cell
  • amphitrichous – one flagellum at each end of cell
  • lophotrichous – cluster of flagella at one or both ends
  • peritrichous – spread over entire surface of cell
  • extends from cell surface to the tip
  • hollow, rigid cylinder
  • composed of the protein flagellin
  • some procaryotes have a sheath around filament
  • filament
flagellar ultrastructure
Flagellar Ultrastructure

The Hook and Basal Body

  • hook
    • links filament to basal body
  • basal body
    • series of rings that drive flagellar motor
flagellar synthesis
Flagellar Synthesis
  • an example of self-assembly
  • complex process involving many genes and gene products
  • new molecules offlagellinare transported through the hollowfilament
  • growth is from tip, not base

Differences of Archaeal Flagella

  • flagella thinner
  • more than one type of flagellin protein
  • flagellum are not hollow
  • hook and basal body difficult to distinguish
  • more related to Type IV secretions systems
  • growth occurs at the base, not the end


Flagellar movement

Spirochete motility

Twitching motility

Gliding motility

  • Bacteria and Archaea have directed movement
  • chemotaxis
    • move toward chemical attractants such as nutrients, away from harmful substances
  • move in response to temperature, light, oxygen, osmotic pressure, and gravity

Bacterial Flagellar Movement

  • flagellum rotates like a propeller
    • very rapid rotation up to 1100 revolutions/sec
    • in general, counterclockwise (CCW) rotation causes forward motion (run)
    • in general, clockwise rotation (CW) disrupts run causing cell to stop and tumble (打滾)

Mechanism of Flagellar Movement

  • flagellum is 2 part motor producing torque (項鍊,轉矩)
  • rotor (轉子)
    • C (FliG protein) ring and MS ring turn and interact with stator
  • stator (發電機的定子;定片)- Mot A and Mot B proteins
    • form channel through plasma membrane
    • protons move through Mot A and Mot B channels and produce energy through proton motive force
    • torque powers rotation of the basal body and filament

Spirochete (螺旋體) Motility

  • multiple flagella form axial fibril which winds around the cell
  • flagella remain in periplasmic space inside outer sheath
  • corkscrew shape exhibits flexing and spinning movements

Twitching (抽動) and Gliding (滑行) Motility

  • may involve Type IV pili and slime
  • twitching
    • pili at ends of cell
    • short, intermittent, jerky motions
    • cells are in contact with each other and surface
  • gliding
    • smooth movements
Chemotaxis (化學趨化作用)
  • movement towards a chemical attractant or away from a chemical repellent
  • concentrations of chemical attractants and chemical repellents detected by chemoreceptors on surfaces of cells
chemotaxis towards attractant
Chemotaxis Towards Attractant
  • in presence of attractant (b) tumbling frequency is reduced and runs in direction of attractant are longer
the bacterial endospore
The Bacterial Endospore
  • formed by some bacteria
  • dormant
  • resistant to numerous environmental conditions
    • heat
    • radiation
    • chemicals
    • desiccation 乾燥

Endospore Structure

  • spore surrounded by thin covering called exosporium
  • thick layers of protein form the spore coat
  • cortex皮層, beneath the coat, thick peptidoglycan
  • core has nucleoid and ribosomes

What makes an endospore so resistant?

  • calcium (complexed with dipicolinic acid)
  • small, acid-soluble, DNA-binding proteins (SASPs)
  • dehydrated core
  • spore coat and exosporium protect
  • Also called endospore formation or sporulation
  • normally commences when growth ceases because of lack of nutrients
  • complex multistage process
germination transformation of dormant spores into active vegetative cells
Germination-Transformation of dormant spores into active vegetative cells
  • activation
    • prepares spores for germination
    • often results from treatments like heating
  • germination
    • spore swelling
    • rupture of absorption of spore coat
    • loss of resistance
    • increased metabolic activity
  • outgrowth
    • emergence of vegetative cell