Microscopy and Cell Structure

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Microscopy and Cell Structure

• Chapter 3

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Microscope TechniquesMicroscopes

• Microscopes
• Most important tool for studying microorganisms
• Use viable light to observe objects
• Magnify images approximately 1,000x
• Electron microscope, introduced in 1931, can
  magnify images in excess of 100,000x
• Scanning probe microscope, introduced in 1981,
  can view individual atoms

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Principles of Light Microscopy

• Light Microscopy
• Light passes through specimen, then through
  series of magnifying lenses
• Most common and easiest to use is the
  bright-field microscope
• Important factors in light microscopy include
• Magnification
• Resolution
• Contrast

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Principles of Light Microscopy

• Magnification
• Microscope has two magnifying lenses
• Called compound microscope
• Lens include
• Ocular lens and objective lens
• Most bright field scopes have four magnifications
  of objective lenses, 4x, 10x, 40x and 100x
• Lenses combine to enlarge objects
• Magnification is equal to the factor of the
  ocular x the objective
• 10x X 100x 1,000x

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Principles of Light Microscopy

• Magnification
• Bright field scopes have condenser lens
• Has no affect on magnification
• Used to focus illumination on specimen

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Principles of Light Microscopy

• Resolution
• Usefulness of microscope depends on its ability
  to resolve two objects that are very close
  together
• Resolving power is defined as the minimum
  distance existing between two objects where those
  objects still appear as separate objects
• Resolving power determines how much detail can be
  seen

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Principles of Light Microscopy

• Resolution
• Resolution depends on the quality of lenses and
  wavelength of illuminating light
• How much light is released from the lens
• Maximum resolving power of most brightfield
  microscopes is 0.2 µm (1x10-6)
• This is sufficient to see most bacterial
  structures
• Too low to see viruses

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Principles of Light Microscopy

• Resolution
• Resolution is enhanced with lenses of higher
  magnification (100x) by the use of immersion oil
• Oil reduces light refraction
• Light bends as it moves from glass to air
• Oil bridges the gap between the specimen slide
  and lens and reduces refraction
• Immersion oil has nearly same refractive index as
  glass

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Principles of Light Microscopy

• Contrast
• Reflects the number of visible shades in a
  specimen
• Higher contrast achieved for microscopy through
  specimen staining

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Principles of Light Microscopy

• Examples of light microscopes that increase
  contrast
• Phase-Contrast Microscope
• Interference Microscope
• Dark-Field Microscope
• Fluorescence Microscope
• Confocal Scanning Laser Microscope

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Principles of Light Microscopy

• Phase-Contrast
• Amplifies differences between refractive indexes
  of cells and surrounding medium
• Uses set of rings and diaphragms to achieve
  resolution

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Principles of Light Microscopy

• Interference Scope
• This microscope causes specimen to appear three
  dimensional
• Depends on differences in refractive index
• Most frequently used interference scope is
  Nomarski differential interference contrast

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Principles of Light Microscopy

• Dark-Field Microscope
• Reverse image
• Specimen appears bright on a dark background
• Like a photographic negative
• Achieves image through a modified condenser

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Bright field vs. Dark field
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Bright field vs. Dark field
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Principles of Light Microscopy

• Fluorescence Microscope
• Used to observe organisms that are naturally
  fluorescent or are flagged with fluorescent dye
• Fluorescent molecule absorbs ultraviolet light
  and emits visible light
• Image fluoresces on dark background

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Principles of Light Microscopy

• Confocal Scanning Laser Microscope
• Used to construct three dimensional image of
  thicker structures
• Provides detailed sectional views of internal
  structures of an intact organism
• Laser sends beam through sections of organism
• Computer constructs 3-D image from sections

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Principles of Light Microscopy

• Electron Microscope
• Uses electromagnetic lenses, electrons and
  fluorescent screen to produce image
• Resolution increased 1,000 fold over brightfield
  microscope
• To about 0.3 nm (1x10-9)
• Magnification increased to 100,000x
• Two types of electron microscopes
• Transmission
• Scanning

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Principles of Light Microscopy

• Transmission Electron Microscope (TEM)
• Used to observe fine detail
• Directs beam of electrons at specimen
• Electrons pass through or scatter at surface
• Shows dark and light areas
• Darker areas more dense
• Specimen preparation through
• Thin sectioning
• Freeze fracturing or freeze etching

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Principles of Light Microscopy

• Scanning Electron Microscope (SEM)
• Used to observe surface detail
• Beam of electrons scan surface of specimen
• Specimen coated with metal
• Usually gold
• Electrons are released and reflected into viewing
  chamber
• Some atomic microscopes capable of seeing single
  atoms

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Microscope TechniquesDyes and Staining

• Dyes and Staining
• Cells are frequently stained to observe organisms
• Satins are made of organic salts
• Dyes carry () or (-) charge on the molecule
• Molecule binds to certain cell structures
• Dyes divided into basic or acidic based on charge
• Basic dyes carry positive charge and bond to cell
  structures that carry negative charge
• Commonly stain the cell
• Acidic dyes carry positive charge and are
  repelled by cell structures that carry negative
  charge
• Commonly stain the background

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Microscope TechniquesDyes and Staining

• Basic dyes () more commonly used than acidic
  dyes (-)
• Common basic () dyes include
• Methylene blue
• Crystal violet
• Safrinin
• Malachite green

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Microscope TechniquesDyes and Staining

• Staining Procedures
• Simple stain uses one basic stain to stain the
  cell
• Allows for increased contrast between cell and
  background
• All cells stained the same color
• No differentiation between cell types

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Microscope TechniquesDyes and Staining

• Differential Stains
• Used to distinguish one bacterial group from
  another
• Uses a series of reagents
• Two most common differential stains
• Gram stain
• Acid-fast stain

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Microscope TechniquesDyes and Staining

• Gram Stain
• Most widely used procedure for staining bacteria
• Developed over century ago
• Dr. Hans Christian Gram
• Bacteria separated into two major groups
• Gram positive
• Stained purple
• Gram negative
• Stained red or pink

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Dyes and Staining

• The Gram Stain

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Gram Positive and Gram Negative Cells
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Microscope TechniquesDyes and Staining

• Acid-fast Stain
• Used to stain organisms that resist conventional
  staining
• Used to stain members of genus Mycobacterium
• High lipid concentration in cell wall prevents
  uptake of dye
• Uses heat to facilitate staining
• Once stained difficult to decolorize

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Microscope TechniquesDyes and Staining

• Acid-fast Stain
• Can be used for presumptive identification in
  diagnosis of clinical specimens
• Requires multiple steps
• Primary dye
• Carbol fuchsin
• Colors acid-fast bacteria red
• Decolorizer
• Generally acid alcohol
• Removes stains from non acid-fast bacteria
• Counter stain
• Methylene blue
• Colors non acid-fast bacteria blue

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The Ziehl-Neesen Acid-Fast Stain
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Microscope TechniquesDyes and Staining

• Special Stains
• Capsule stain
• Example of negative stain
• Allows capsule to stand out around organism
• Endospore stain
• Staining enhances endospore
• Uses heat to facilitate staining
• Flagella stain
• Staining increases diameter of flagella
• Makes more visible

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Morphology of Prokaryotic Cells

• Prokaryotes exhibit a variety of shapes
• Most common
• Coccus
• Spherical
• Bacillus
• Rod or cylinder shaped
• Cell shape not to be confused with Bacillus genus

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Morphology of Prokaryotic Cells

• Prokaryotes exhibit a variety of shapes
• Other shapes
• Coccobacillus
• Short round rod
• Vibrio
• Curved rod
• Spirillum
• Spiral shaped
• Spirochete
• Helical shape
• Pleomorphic
• Bacteria able to vary shape

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Morphology of Prokaryotic Cells

• Prokaryotic cells may form groupings after cell
  division
• Cells adhere together after cell division for
  characteristic arrangements
• Arrangement depends on plan of division
• Especially in the cocci

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Morphology of Prokaryotic Cells

• Division along a single plane may result in pairs
  or chains of cells
• Pairs diplococci
• Example Neisseria gonorrhoeae
• Chains streptococci
• Example species of Streptococcus

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Morphology of Prokaryotic Cells

• Division along two or three perpendicular planes
  form cubical packets
• Example Sarcina genus
• Division along several random planes form
  clusters
• Example species of Staphylococcus

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Morphology of Prokaryotic Cells

• Some bacteria live in groups with other bacterial
  cells
• They form multicellular associations
• Example myxobacteria
• These organisms form a swarm of cells
• Allows for the release of enzymes which degrade
  organic material
• In the absence of water cells for fruiting bodies
• Other organisms for biofilms
• Formation allows for changes in cellular activity

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Cytoplasmic Membrane

• Cytoplasmic membrane
• Delicate thin fluid structure
• Surrounds cytoplasm of cell
• Defines boundary
• Serves as a semi permeable barrier
• Barrier between cell and external environment

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Cytoplasmic Membrane

• Structure is a lipid bilayer with embedded
  proteins
• Bilayer consists of two opposing leaflets
• Leaflets composed of phospholipids
• Each contains a hydrophilic phosphate head and
  hydrophobic fatty acid tail

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The Basic Structural Component of the Membrane
Phospholipid Molecule
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Cytoplasmic Membrane

• Membrane is embedded with numerous protein
• More that 200 different proteins
• Proteins function as receptors and transport
  gates
• Provides mechanism to sense surroundings
• Proteins are not stationary
• Constantly changing position
• Called fluid mosaic model

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The Fluid-Mosaic Model of the Membrane Structure
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Cytoplasmic Membrane

• Cytoplasmic membrane is selectively permeable
• Determines which molecules pass into or out of
  cell
• Few molecules pass through freely
• Molecules pass through membrane via simple
  diffusion or transport mechanisms that may
  require carrier proteins and energy

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Cytoplasmic Membrane

• Simple diffusion
• Process by which molecules move freely across the
  cytoplasmic membrane
• Water, certain gases and small hydrophobic
  molecules pass through via simple diffusion

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Cytoplasmic Membrane

• Simple diffusion
• Osmosis
• The ability of water to flow freely across the
  cytoplasmic membrane
• Water flows to equalize solute concentrations
  inside and outside the cell
• Inflow of water exerts osmotic pressure on
  membrane
• Membrane rupture is prevented by rigid cell wall
  of bacteria

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Cytoplasmic Membrane

• Membrane also the site of energy production
• Energy produced through series of embedded
  proteins
• Electron transport chain
• Proteins are used in the formation of proton
  motive force
• Energy produced in proton motive force is used to
  drive other transport mechanisms

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Cytoplasmic Membrane

• Directed movement across the membrane
• Movement of many molecules directed by transport
  systems
• Transport systems employ highly selective
  proteins
• Transport proteins (a.k.a permeases or carriers)
• These proteins span membrane
• Single carrier transports specific type molecule
• Most transport proteins are produced in response
  to need
• Transport systems include
• Facilitated diffusion
• Active transport
• Group translocation

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Cytoplasmic Membrane

• Facilitated diffusion
• Moves compounds across membrane exploiting a
  concentration gradient
• Flow from area of greater concentration to area
  of lesser concentration
• Molecules are transported until equilibrium is
  reached
• System can only eliminate concentration gradient
  it cannot create one
• No energy is required for facilitated diffusion
• Example movement of glycerol into the cell

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Cytoplasmic Membrane

• Active transport
• Moves compounds against a concentration gradient
• Requires an expenditure of energy
• Two primary mechanisms
• Proton motive force
• ATP Binding Cassette system

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Cytoplasmic Membrane

• Proton motive force
• Transporters allow protons into cell
• Protons either bring in or expel other substances
• Example efflux pumps used in antimicrobial
  resistance
• ATP Binding Cassette system (ABC transport)
• Use binding proteins to scavenge and deliver
  molecules to transport complex
• Example maltose transport

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Cytoplasmic Membrane

• Group transport
• Transport mechanism that chemically alters
  molecule during passage
• Uptake of molecule does not alter concentration
  gradient
• Phosphotransferase system example of group
  transport mechanism
• Phosphorylates sugar molecule during transport
• Phosphorylation changes molecule and therefore
  does not change sugar balance across the membrane

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Cell Wall

• Bacterial cell wall
• Rigid structure
• Surrounds cytoplasmic membrane
• Determines shape of bacteria
• Holds cell together
• Prevents cell from bursting
• Unique chemical structure
• Distinguishes Gram positive from Gram-negative

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Cell Wall

• Rigidity of cell wall is due to peptidoglycan
  (PTG)
• Compound found only in bacteria
• Basic structure of peptidoglycan
• Alternating series of two subunits
• N-acetylglucosamin (NAG)
• N-acetylmuramic acid (NAM)
• Joined subunits form glycan chain
• Glycan chains held together by string of four
  amino acids
• Tetrapeptide chain

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Cell Wall

• Gram positive cell wall
• Relatively thick layer of PTG
• As many as 30
• Regardless of thickness, PTG is permeable to
  numerous substances
• Teichoic acid component of PTG
• Gives cell negative charge

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TYPICAL PROKARYOTIC CELL
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Gram Positive Bacterial Cell Wall
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Gram Negative Bacterial Cell Wall
Note thin Peptidoglycan layer inside a
Lipopolysaccharide layer
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Cell Wall

• Gram-negative cell wall
• More complex than G
• Only contains thin layer of PTG
• PTG sandwiched between outer membrane and
  cytoplasmic membrane
• Region between outer membrane and cytoplasmic
  membrane is called periplasm
• Most secreted proteins contained here
• Proteins of ABC transport system located here

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Cell Wall

• Outer membrane
• Constructed of lipid bilayer
• Much like cytoplasmic membrane but outer leaflet
  made of lipopolysaccharides not phospholipids
• Outer membrane also called the lipopolysaccharide
  layer or LPS layer
• LPS severs as barrier to a large number of
  molecules
• Small molecules or ions pass through channels
  called porins
• Portions of LPS medically significant
• O-specific polysaccharide side chain
• Lipid A

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Cell Wall

• O-specific polysaccharide side chain
• Directed away from membrane
• Opposite location of Lipid A
• Used to identify certain species or strains
• E. coli O157H7 refers to specific O-side chain
• Lipid A
• Portion that anchors LPS molecule in lipid
  bilayer
• Plays role in recognition of infection
• Molecule present with Gram negative infection of
  bloodstream

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Cell Wall

• Peptidoglyan (PTG) as a target
• Many antimicrobial interfere with the synthesis
  of PTG
• Examples include
• Penicillin
• Lysozyme

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Cell Wall

• Penicillin
• Binds proteins involved in cell wall synthesis
• Prevents cross-linking of glycan chains by
  tetrapeptides
• More effective against Gram positive bacterium
• Due to increased concentration of PTG
• Penicillin derivatives produced to protect
  against Gram negatives

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Cell Wall

• Lysozymes
• Produced in many body fluids including tears and
  saliva
• Breaks bond linking NAG and NAM
• Destroys structural integrity of cell wall
• Enzyme often used in laboratory to remove PTG
  layer from bacteria
• Produces protoplast in G bacteria
• Produces spheroplast in G- bacteria

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Cell Wall

• Differences in cell wall account for differences
  in staining characteristics
• Gram-positive bacterium retain crystal
  violet-iodine complex of Gram stain
• Gram-negative bacterium lose crystal
  violet-iodine complex

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Cell Wall

• Some bacterium naturally lack cell wall
• Mycoplasma
• Bacterium causes mild pneumonia
• Have no cell wall
• Antimicrobial directed towards cell wall
  ineffective
• Sterols in membrane account for strength of
  membrane
• Bacteria in Domain Archaea
• Have a wide variety of cell wall types
• None contain peptidoglycan but rather
  pseudopeptidoglycan

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Layers External to Cell Wall

• Capsules and Slime Layer
• General function
• Protection
• Protects bacteria from host defenses
• Attachment
• Enables bacteria to adhere to specific surfaces
• Capsule is a distinct gelatinous layer
• Slime layer is irregular diffuse layer
• Chemical composition of capsules and slime layers
  varies depending on bacterial species
• Most are made of polysaccharide
• Referred to as glycocalyx
• Glyco sugar calyx shell

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Flagella and Pili

• Some bacteria have protein appendages
• Not essential for life
• Aid in survival in certain environments
• They include
• Flagella
• Pili

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Flagella and Pili

• Flagella
• Long protein structure
• Responsible for motility
• Use propeller like movements to push bacteria
• Can rotate more than 100,00 revolutions/minute
• 82 mile/hour
• Some important in bacterial pathogenesis
• H. pylori penetration through mucous coat

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Flagella and Pili

• Flagella structure has three basic parts
• Filament
• Extends to exterior
• Made of proteins called flagellin
• Hook
• Connects filament to cell
• Basal body
• Anchors flagellum into cell wall

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Flagella and Pili

• Bacteria use flagella for motility
• Motile through sensing chemicals
• Chemotaxis
• If chemical compound is nutrient
• Acts as attractant
• If compound is toxic
• Acts as repellent
• Flagella rotation responsible for run and tumble
  movement of bacteria

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CHEMOTAXIS
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Flagella and Pili

• Pili
• Considerably shorter and thinner than flagella
• Similar in structure
• Protein subunits
• Function
• Attachment
• These pili called fimbre
• Movement
• Conjugation
• Mechanism of DNA transfer

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Internal Structures

• Bacterial cells have variety of internal
  structures
• Some structures are essential for life
• Chromosome
• Ribosome
• Others are optional and can confer selective
  advantage
• Plasmid
• Storage granules
• Endospores

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Internal Structures

• Chromosome
• Resides in cytoplasm
• In nucleoid space
• Typically single chromosome
• Circular double-stranded molecule
• Contains all genetic information
• Plasmid
• Circular DNA molecule
• Generally 0.1 to 10 size of chromosome
• Extrachromosomal
• Independently replicating
• Encode characteristic
• Potentially enhances survival
• Antimicrobial resistance

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Internal Structure

• Ribosome
• Involved in protein synthesis
• Composed of large and small subunits
• Units made of riboprotein and ribosomal RNA
• Prokaryotic ribosomal subunits
• Large 30S
• Small 50S
• Total 70S
• Larger than eukaryotic ribosomes
• 40S, 60S, 80S
• Difference often used as target for antimicrobials

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Internal Structures

• Storage granules
• Accumulation of polymers
• Synthesized from excess nutrient
• Example glycogen
• Excess glucose in cell is stored in glycogen
  granules
• Gas vesicles
• Small protein compartments
• Provides buoyancy to cell
• Regulating vesicles allows organisms to reach
  ideal position in environment

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Internal Structures

• Endospores
• Dormant cell types
• Produced through sporulation
• Theoretically remain dormant for 100 years
• Resistant to damaging conditions
• Heat, desiccation, chemicals and UV light
• Vegetative cell produced through germination
• Germination occurs after exposure to heat or
  chemicals
• Germination not a source of reproduction

Common bacteria genus that produce endospores
include Clostridium and Bacillus
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The Schaeffer-Fulton Spore Stain
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Internal Structures

• Endospore formation
• Complex, ordered sequence
• Bacteria sense starvation and begin sporulation
• Growth stops
• DNA duplicated
• Cell splits
• Cell splits unevenly
• Larger component engulfs small component,
  produces forespore within mother cell
• Forespore enclosed by two membranes
• Forespore becomes core
• PTG between membranes forms core wall and cortex
• Mother cell proteins produce spore coat
• Mother cell degrades and releases endospore

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Endospore
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Eukaryotic Plasma Membrane

• Similar in chemical structure and function of
  cytoplasmic membrane of prokayote
• Phospholipid bilayer embedded with proteins
• Proteins in bilayer perform specific functions
• Transport
• Maintain cell integrity
• Attachment of proteins to internal structures
• Receptors for cell signaling
• Proteins in outer layer
• Receptors typically glycoproteins
• Membrane contains sterols for strength
• Animal cells contain cholesterol
• Fungal cells contain ergosterol
• Difference in sterols target for antifungal
  medications

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Eukaryotic Plasma Membrane

• Transport across eukaryotic membrane
• Some molecules pass through membrane via
  transport proteins
• Others taken in through endocytosis and exocytosis

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Eukaryotic Plasma Membrane

• Transport proteins
• Function as carriers or channels
• Channels create pores in membrane
• Channels are gated
• Open or closed depending on environmental
  conditions
• Concentration gradient
• Carriers analogous to prokaryotic membrane
  proteins
• Mediate facilitated diffusion and active transport

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Eukaryotic Plasma Membrane

• Endocytosis
• Process by which eukaryotic cells bring in
  material from surrounding environment
• Pinocytosis most common type in animal cell
• Pinch off small portions of own membrane along
  with attached material
• Internalize vesicle and contents
• Vesicle called endosome

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Eukaryotic Plasma Membrane

• Endocytosis
• Phagocytosis
• Specific type of endocytosis
• Important in body defenses
• Phagocyte sends out pseudopods to surround
  microbes
• Phagocyte brings microbe into vacuole
• Vacuole phagosome
• Phagosome fuses with a sack of enzymes and toxins
• Sack lysosome
• Fusion of phagosome and lysosome creates
  phagolysosome
• Microbe dies in phagolysosome
• Phagosome breaks down microbial material

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Eukaryotic Plasma Membrane

• Exocytosis
• Reverse of endocytosis
• Vesicles inside cell fuse with plasma membrane
• Releases contents into external environment

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Protein Structures of Eukaryotic Cell

• Eukaryotic cells have unique structures that
  distinguish them from prokaryotic
• Cytoskeleton
• Flagella
• Cilia
• 80s ribosome

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Protein Structures of Eukaryotic Cell

• Cytoskeleton
• Threadlike proteins
• Reconstructs to adapt to cells changing needs
• Composed of three elements
• Microtubules
• Actin filaments
• Intermediate fibers

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Protein Structures of Eukaryotic Cell

• Microtubules
• Thickest of cytoskeleton structures
• Long hollow cylinders
• Protein subunits called tubulin
• Form mitotic spindles
• Main structures in cilia and flagella

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Protein Structures of Eukaryotic Cell

• Actin filaments
• Composed of actin polymer
• Enable cell cytoplasm to move
• Assembles and disassembles causing motion
• Pseudopod formation

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Protein Structures of Eukaryotic Cell

• Intermediate fibers
• Function to strengthen cell
• Enable cells to resist physical stress

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Protein Structures of Eukaryotic Cell

• Flagella
• Flexible structure
• Function in motility
• 92 arrangement
• 9 pairs of microtubules surrounded by 2
  individual
• Cilia
• Shorter than flagella
• Often cover cell
• Can move cell or propel surroundings along
  stationary cell

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Flagella
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Arrangements of Bacterial Flagella

• Monotrichous Bacteria with a single polar
  flagellum located at one end (pole)
• Amphitrichous Bacteria with two flagella, one
  at each end
• Peritrichous Bacteria with flagella all over the
  surface
• Atrichous Bacteria without flagella
• Cocci shaped bacteria rarely have flagella

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Polar, monotrichous flagellum
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Polar, amphitrichous flagellum
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Peritrichous flagella
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Proteus (29,400X)
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Membrane-bound Organellesof Eukaryotes

• Eukaryotes have numerous organelles that set them
  apart from prokaryotic cells
• Nucleus
• Mitochondria and chloroplast
• Endoplasmic reticulum
• Golgi apparatus
• Lysosome and peroxisomes

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Membrane-bound Organellesof Eukaryotes

• Nucleus
• Distinguishing feature of eukaryotic cell
• Contains DNA
• Area of DNA replication
• Mitosis asexual
• Meiosis sexual
• Mitochondria
• Site of energy production
• Surrounded by membrane bilayer
• Inner and outer membrane
• Outer membrane invaginations called cristae
• Matrix formed from inner membrane
• Contains DNA

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Membrane-bound Organellesof Eukaryotes

• Chloroplast
• Found only in plant and algae
• Site of photosynthesis
• Surrounded by two membranes
• Endoplasmic reticulum
• Divided into rough and smooth
• Rough ER
• embedded with ribosomes
• Site of protein synthesis
• Smooth ER
• Lipid synthesis and degradation
• Calcium storage

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Membrane-bound Organellesof Eukaryotes

• Golgi apparatus
• Consists of a series of membrane bound flattened
  sacs
• Modifies macromolecules produced in endoplasmic
  reticulum
• Lysosomes Peroxisomes
• Lysosomes contain degradative enzymes
• Proteases and nucleases
• Peroxisomes
• Organelles in which oxygen is used to oxidize
  substances
• Breaking down lipids
• detoxification