A Brief Journey to the Microbial World

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Chapter 2

• A Brief Journey to the Microbial World

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Microscopy

• Microscopes are essential for microbiological
  studies.
• A microscope is required for the visualization of
  microorganisms
• Light microscopy is used to observe less detailed
  features of intact cell under low magnification
  than advanced microscopy (i.e. electron and
  laser)
• Various types of light microscopes exist,
  including bright-field, dark-field, phase
  contrast, and fluorescence microscopes.

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Magnification vs. Resolution

• Magnification
• Increase in apparent size
• Two sets of lenses form the image
• Objective lens and ocular lens
• Total magnification is a product of the
  magnification of the two sets of lenses
• Objective magnification X ocular magnification
• Resolution clarity
• Ability to see 2 nearby objects as distinct
  objects
• All compound light microscopes optimize image
  resolution by using lenses with high
  light-gathering characteristics (numerical
  aperture).
• The limit of resolution for a light microscope is
  about 0.2 ?m.

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Microscopy
Figure 2.1b
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Simple and/or differential cell staining are used
to increase contrast in bright-field microscopy.

• Improving contrast results in a better final
  image
• Staining is an easy way to improve contrast
• Dyes are organic compounds that have different
  affinities for specific cellular materials
• Examples of common stains are methylene blue,
  safranin, and crystal violet

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Staining Cells for Microscopic Observation
Figure 2.3
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Staining Cells for Microscopic Observation
Figure 2.3
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Staining Cells for Microscopic Observation
Figure 2.3
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Differential StainsThe Gram stain

• The Gram stain is widely used in microbiology
• On the basis of the Gram stain, bacteria can be
  divided into two major groups gram-positive and
  gram-negative
• The Gram stain renders different kinds of cells
  different colors
• Gram-positive bacteria appear purple and
  gram-negative bacteria appear pink to red after
  staining

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The Gram Stain Steps in the Gram-stain Procedure
Figure 2.4a
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The Gram Stain Steps in the Gram-stain Procedure
Figure 2.4a
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The Gram Stain
Figure 2.4b and c
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Phase Contrast Microscopy

• Invented in 1936 by Frits Zernike
• May be used to visualize live samples and avoid
  distortion from cell stains
• Can see some internal features
• Resulting image is dark cells on a light
  background

Figure 2.5
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Electron Microscopy

• Electron microscopes have far greater resolving
  power than light microscopes, with limits of
  resolution of about 0.2 nm.
• Electron microscopes use electrons instead of
  photons to image cells and structures
• Two types
• Transmission electron microscopy (TEM)
• For observing internal cell structure down to the
  molecular level
• Scanning electron microscopy (SEM)
• For three-dimensional imaging and examining
  surfaces

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Electron Micrographs
Figure 2.10a
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Electron Micrographs
Figure 2.10bc
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All microbial cells share certain basic
structures in common

• Cytoplasm
• Cytoplasmic membrane
• Allows passage of needed molecules (nutrients,
  water, etc.) and barrier to harmful chemicals
• Ribosomes
• Site of protein synthesis
• Cell wall (usually)
• Cell shape

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Prokaryote vs Eukaryote

• Two structural types of cells are recognized
• Prokaryotic
• Archaea and bacteria
• Eukaryotic plants, algae, fungi, protists, and
  animals (variety)
• Comparing prokaryotic and eukaryotic cells
• Prokaryote comes from the Greek words for
  prenucleus.
• Eukaryote comes from the Greek words for true
  nucleus.

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Prokaryote Eukaryote

• Simpler internal structure
• Absence of nucleus
• One circular chromosome, not in a membrane
• No histones
• No membrane enclosed organelles
• Peptidoglycan cell walls
• Binary fission for cell division
• Smaller

• Contain nucleus
• Paired chromosomes, in nuclear membrane
• Histones
• Membrane enclosed organelles
• Simple (polysaccharide) cell walls
• Cell division by mitosis or meiosis
• Larger

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Structure of Prokaryotic vs. Eukaryotic cell
Figure 2.11a
Figure 2.11b
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Viruses

• Non cellular
• Obligate intracellular parasites
• They must live inside another cell to survive
• Have only one type of nucleic acid
• DNA or RNA (never both)
• Single or Double stranded
• Protein coat (no plasma membrane)
• Few to no enzymes
• Takes enzymes and use host cell metabolic
  machinery
• No metabolic activity
• They require a host cell to exhibit the
  characteristics of life.
• Virus diversity
• Different viruses have different hosts
• Only some viruses cause disease

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Size

• Typical prokaryote ?1 - 5 ?m long
• Typical Eukaryotic cell ?10 - 100 ?m
• Typical virus ? 50 - 80 nm

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Phylogeny

• The study of the evolutionary relationionships of
  distinct organisms
• Although species of Bacteria and Archaea share a
  prokaryotic cell structure, they differ
  dramatically in their evolutionary history.
• Molecular based
• Compare sequences from common molecules from
  organisms of interest
• Relationships can be deduced by comparing genetic
  information (nucleic acid or amino acid
  sequences) in the different specimens
• Carl Woese (1970s)
• rRNA comparison
• Ribosomal RNA (rRNA) are excellent molecules for
  determining phylogeny
• Can visualize relationships on a phylogenetic
  tree

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Ribosomal RNA (rRNA) Gene Sequencing and Phylogeny
Figure 2.16
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Phylogeny

• Comparative ribosomal RNA sequencing has defined
  the three domains of life
• Bacteria (prokaryotic)
• Archaea (prokaryotic)
• Eukarya (eukaryotic)
• Common ancestor - over 3.8 billion years ago
• Bacteria and archaea are prokaryotes but archaea
  are more closely related to eukaryotes
• Eukaryotic microorganisms were the ancestors of
  multicellular organisms
• Mitochondria and chloroplasts also contain their
  own genomes (circular, like prokaryotes) and
  ribosomes
• These organelles are ancestors of specific
  lineages of Bacteria
• Mitochondria and chloroplasts took up residence
  in Eukarya eons ago
• This arrangement is known as endosymbiosis

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The Tree of Life Defined by rRNA Sequencing
Figure 2.17
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2.8 - Physiological Diversity of Microorganisms

• The phylogenetic diversity we see in microbial
  cells is the product of almost 4 billion years of
  evolution
• Microorganisms also have a tremendous amount of
  metabolic diversity
• Microorganisms have exploited every conceivable
  means of making a living consistent with the
  laws of chemistry and physics

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Physiological Diversity

• All cells need carbon and energy sources.
• Different species use different strategies
• Chemoorganotrophs obtain their energy from the
  oxidation of organic compounds.
• Chemolithotrophs obtain their energy from the
  oxidation of inorganic compounds.
• Phototrophs contain pigments that allow them to
  use light as an energy source.
• Oxygenic versus anoxygenic photosynthesis

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Metabolic Options for Conserving Energy
Figure 2.18
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Autotrophs vs. Heterotrophs

• All cells require carbon as a major nutrient
• Autotrophs
• Use carbon dioxide as their carbon source
• Use sunlight for energy
• Primary producers
• Most phototrophs and lithotrophs are autotrophs
• Heterotrophs
• Use organic carbon as their carbon source
• Use products of autotrophs or the autotrophs
  themselves for energy
• All organotrophs and SOME lithotrophs are
  heterotrophs

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Extremophiles

• Thrive under environmental conditions in which
  higher organisms cannot survive.
• Prokaryotes thrive in habitats that are too cold,
  too hot, too salty, too basic for any eukaryote
• Many prokaryotes are extremophiles
• No environment is devoid of prokaryotic life
• Salt concentrations up to 30 for some
• halophiles
• pH 0-12
• Acidophiles and alkaliphiles
• Temps below 0 ºC to above 100 ºC
• Psychrophiles and hyperthermophiles
• High pressure
• Barophiles

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Classes and Examples of Extremophiles