Microscopy and Cell Structure

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Microscopy and Cell Structure

• Chapter 3
• Part I Observing Cells

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Microscope TechniquesMicroscopes
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Principles of Light Microscopy

• Light Microscopy
• Most common and easiest to use bright-field
  microscope
• Important factors in light microscopy include
• Magnification
• Resolution
• Contrast

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Principles of Light Microscopy

• Magnification
• two magnifying lenses
• Ocular lens and objective lens
• condenser lens
• focus illumination on specimen

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Principles of Light Microscopy

• Resolution
• minimum distance between two objects that still
  appear as separate objects
• determine the usefulness of microscope

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Principles of Light Microscopy

• Factors affect resolution
• Lens
• Wavelength of light
• How much light is released from the lens
• magnification
• Maximum resolving power of most brightfield
  microscopes is 0.2 µm (1x10-6)
• sufficient to see most bacteria
• Too low to see viruses

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Principles of Light Microscopy

• Resolution is enhanced with lenses of higher
  magnification (100x) by the use of immersion oil
• Oil reduces light 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
• increase contrast
• Use special microscopes
• 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
• Darker appearance for denser materials.
• Uses set of rings and diaphragms to achieve
  resolution

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Principles of Light Microscopy

• Interference Scope
• appear three dimensional
• Depends on differences in refractive index

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Principles of Light Microscopy

• Dark-Field Microscope
• Reverse image
• Like a photographic negative
• a modified condenser directs the lights at an
  angle and only the light scattered by the
  specimen enters the objective lens

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Principles of Light Microscopy

• Fluorescence Microscope
• observe organisms naturally fluorescent or
  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

• 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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Quiz

• With 10x ocular lens and 40x objective lens, what
  is the magnifying power?

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Quiz

• What are the three important factors for
  microscope?

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Microscope TechniquesDyes and Staining

• Dyes and Staining
• stained to observe organisms
• made of organic salts
• Basic dyes carry positive charge
• Acidic dyes carry negative charge

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Microscope TechniquesDyes and Staining

• Common basic dyes include
• Methylene blue
• Crystal violet
• Safrinin
• Malachite green

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Microscope TechniquesDyes and Staining

• Simple staining
• use one color to stain
• increase contrast between cell and background

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Microscope TechniquesDyes and Staining

• Differential Stains
• 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
• widely used procedure for classiffying bacteria
• two major groups based on cell wall structural
  differences
• Gram positive
• Gram negative

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Microscope TechniquesDyes and Staining

• Gram Stain
• Involves four reagents
• Primary stain
• Mordent
• Decolorizer
• Counter or Secondary stain

Old gram positive appears to be gram negative
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Microscope TechniquesDyes and Staining

• Acid-fast Stain
• Used to stain members of genus Mycobacterium
• High lipid concentration in cell wall
• Uses heat to facilitate staining

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Microscope TechniquesDyes and Staining

• Acid-fast Stain
• used for presumptive identification in diagnosis
  of clinical specimens
• Requires multiple steps
• Primary dye
• Decolorizer
• Counter stain

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Microscope TechniquesDyes and Staining

• Special Stains
• Capsule stain
• Endospore stain
• Uses heat to facilitate staining
• Flagella stain

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Quiz

• What are the two commonly used differential
  staining method?

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Morphology of Prokaryotic Cells

• Prokaryotes exhibit a variety of shapes
• Coccus
• Bacillus
• Do not to be confused with Bacillus genus

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Morphology of Prokaryotic Cells

• Coccobacillus
• Vibrio
• Spirillum
• Spirochete
• Pleomorphic

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Morphology of Prokaryotic Cells

• groupings morphology
• Cells adhere together after cell division for
  characteristic arrangements
• 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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Review of Chapter III part I
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Microscope

• Three important factors
• Staining.
• Prokaryotic morphology

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Microscopy and Cell Structure

• Part II - Prokaryotic Cell Structure

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Cytoplasmic membrane

• Defines the boundary of the cell

• Semi-permeable

• Transport proteins function as selective gates
  (selectively permeable)
• Control entrance/expulsion of antimicrobial drugs
• Receptors provide a sensor system

• Phospholipid bilayer, embedded with proteins

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Cytoplasmic membrane

• Defines the boundary of the cell

• Semi-permeable

• Transport proteins function as selective gates
  (selectively permeable)
• Control entrance/expulsion of antimicrobial drugs
• Receptors provide a sensor system

• Phospholipid bilayer, embedded with proteins

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Cytoplasmic membrane

• Defines the boundary of the cell

• Semi-permeable

• Transport proteins function as selective gates
  (selectively permeable)
• Control entrance/expulsion of antimicrobial drugs
• Receptors provide a sensor system

• Phospholipid bilayer, embedded with proteins

• Fluid mosaic model

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Cytoplasmic Membrane

• Methods for molecule to go cross membrane
• Simple diffusion the only system does not rely
  on transport protein
• Facilitated diffusion
• Active transport
• Group transport

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Cytoplasmic Membrane

• Simple diffusion-
• Water, certain gases and small hydrophobic
  molecules
• Move along with concentration gradient
• Osmosis

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Cytoplasmic Membrane

• Movement of molecules across membrane by
  transport systems
• Specific
• Transport systems include
• Facilitated diffusion
• Active transport
• Group translocation

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Directed Movement of Molecules Across the
Cytoplasmic Membrane
Facilitated diffusion
no energy expended
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Directed Movement of Molecules Across the
Cytoplasmic Membrane
Facilitated diffusion Active transport
- energy is expended
Moves compounds against a concentration gradient
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Directed Movement of Molecules Across the
Cytoplasmic Membrane
Facilitated diffusion Active transport
- energy is expended
Use binding proteins to scavenge and deliver
molecules to transport complex Example maltose
transport
Example efflux pumps used in antimicrobial
resistance
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Cytoplasmic membrane
Proton H
Proton motive force Energy stored in the
electrochemical gradient created by electron
transport chain
Electron transport chain - Series of proteins
that sequentially transfer electrons and eject
protons from the cell, creating an
electrochemical gradient
Electron transport chain

• Proton motive force is used to fuel
• Synthesis of ATP (the cells energy currency)
• Rotation of flagella (motility)
• One form of transport

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Directed Movement of Molecules Across the
Cytoplasmic Membrane
Facilitated diffusion Active transport
- Chemically modifies a compound during transport
Group translocation
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Directed Movement of Molecules Across the
Cytoplasmic Membrane
Facilitated diffusion Active transport Group
translocation Secretion
- Transport of proteins to the outside
Characteristic sequence of amino acids in a newly
synthesized protein functions as a tag (signal
sequence)
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Prokaryotic structure

• Cell membrane structure
• Movements across membrane

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Cell Wall
Provides rigidity to the cell (prevents it from
bursting)
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Cell Wall

• Bacterial cell wall
• Rigid structure
• Determines shape of bacteria
• Protection
• Unique chemical structure
• Distinguishes Gram positive from Gram-negative

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Cell Wall

• Peptidoglycan - rigid molecule unique to bacteria

• Alternating subunits of NAG and NAM form glycan
  chains

• Glycan chains are connected to each other via
  peptide chains on NAM molecules

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Cell Wall
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Cell Wall

• Peptidoglycan - rigid molecule unique to bacteria

• Alternating subunits of NAG and NAM form glycan
  chains

• Glycan chains are connected to each other via
  peptide chains on NAM molecules
• Gram negativedirect join
• Gram positivepeptide interbridge

• Medical significance of peptidoglycan
• Target for selective toxicity synthesis is
  targeted by certain antimicrobial medications
  (penicillins, cephalosporins)
• Recognized by innate immune system
• Target of lysozyme (in egg whites, tears)

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Cell Wall Gram-positive
Thick layer of peptidoglycan Teichoic acids
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Cell WallGram-negative
Thin layer of peptidoglycan Outer membrane -
additional membrane barrier porins permit
passage lipopolysaccharide (LPS)
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Cell WallGram-negative
Thin layer of peptidoglycan Outer membrane -
additional membrane barrier porins permit
passage lipopolysaccharide (LPS)
- ex. E. coli O157H7
endotoxin
- recognized by innate immune system
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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 growing Gram positive
  bacterium
• 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. More effective on gram .
• Produces protoplast in G bacteria
• Produces spheroplast in G- bacteria

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Cell Wall

• Some bacterium naturally lack cell wall
• Mycoplasma
• causes mild pneumonia
• Naturally resistant to penicillin
• 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
• Capsule is a distinct gelatinous layer
• Slime layer is irregular diffuse layer
• polysaccharide
• functions
• Protection
• Attachment
• Biofilm
• Dental plaque

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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
• propeller movements
• more than 100,000 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
• Chemotaxis
• attractant, repellent
• Tumble, run

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Flagella and Pili

• Pili
• shorter and thinner
• Similar in structure
• Protein subunits
• Function
• Attachment
• Movement (jerky movement or glide)
• Conjugation
• Mechanism of DNA transfer (F pili)

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Review for external structure

• Cell membrane component
• Transportation across membrane
• Cell wall structure
• Gram positive
• Gram negative
• Drugs targeting cell wall
• Capsule/slime layer function
• Flagella/pili function

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Internal Structures
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Internal Structures

• Some are essential for life
• Chromosome
• Ribosome
• Others 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
• Potentially enhances survival

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Internal Structure

• Ribosome
• protein synthesis
• large and small subunits
• riboprotein and ribosomal RNA
• Prokaryotic ribosomal subunits
• Large 50S
• Small 30S
• Total 70S
• Smaller 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 granules
• Gas vesicles
• Small protein compartments

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Internal Structures

• Endospores
• Dormant cell types
• Produced through sporulation
• Can survive for long time
• 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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Internal Structures

• Endospore formation
• Bacteria sense starvation and begin sporulation,
  growth stops
• DNA duplicated
• Cell splits unevenly
• Forespore becomes core
• PTG between membranes forms core wall and cortex
• Mother cell proteins produce spore coat
• Mother cell degrades and releases endospore

NOT a method of reproduction One cell ? one
endospore ? one cell
(sporulation)
(germination)
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Microscopy and Cell Structure

• Part III - Eukaryotic Cell Structure
• A BRIEF overview

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Membrane-bound organelles
Animal cell
Plant cell
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Eukaryotic Plasma Membrane

• Similar in chemical structure and function to
  prokayote
• Proteins in bilayer perform specific functions
• 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
• Transport proteins (function as carriers or
  channels)
• Carriers analogous to prokaryotic membrane
  proteins
• Channels Gated pores in membrane.
• Open or closed depending on environmental
  conditions
• Move with concentration gradient
• Some depend on endocytosis and exocytosis

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Eukaryotic Plasma Membrane

• Endocytosis
• Process by which eukaryotic cells bring in
  material from surrounding environment
• Pinocytosis
• Phagocytosis

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Eukaryotic Plasma Membrane

• Phagocytosis
• Important in body defenses
• Phagocyte sends out pseudopods to surround
  microbes
• Phagosome fuses with lysosome and creates
  phagolysosome
• Phagolysosome 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
• 40s 60s

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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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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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Organelles of note Mitochondria and Chloroplasts

• DNA
• ribosomes

(70S)

• DNA sequences similar to rickettsias

Endosymbiotic theory - Perspective 3.1, p. 76

• DNA
• 70S ribosomes
• DNA sequences similar to cyanobacteria

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Membrane-bound Organellesof Eukaryotes

• Nucleus
• Distinguishing feature of eukaryotic cell
• Two lipid bilayers
• Contains chromosomal DNA (linear)
• Area of DNA replication

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Membrane-bound Organellesof Eukaryotes

• Endoplasmic reticulum
• Divided into rough and smooth
• Rough ER
• Smooth ER

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Membrane-bound Organellesof Eukaryotes

• Golgi apparatus
• a series of membrane bound flattened sacs
• Modifies macromolecules produced in endoplasmic
  reticulum
• Lysosomes
• Peroxisomes

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