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Microscopy and Cell Structure
Chapter 3
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
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
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
Magnification Bright field scopes have condenser lens
Has no affect on magnification Used to focus illumination on specimen
Principles of Light Microscopy
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
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
Principles of Light Microscopy
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
Contrast Reflects the number of visible shades in a
specimen Higher contrast achieved for microscopy
through specimen staining
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
Principles of Light Microscopy
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
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
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
Bright field vs. Dark field
Bright field vs. Dark field
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
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
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
Principles of Light Microscopy
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
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
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
Microscope TechniquesDyes and Staining
Basic dyes (+) more commonly used than acidic dyes (-)
Common basic (+) dyes include Methylene blue Crystal violet Safrinin Malachite green
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
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
Microscope TechniquesDyes and Staining
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
Dyes and Staining The Gram Stain
Gram Positive and Gram Negative Cells
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
Microscope TechniquesDyes and Staining
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
The Ziehl-Neesen Acid-Fast Stain
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
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
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
Morphology of Prokaryotic Cells
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
Division along a single plane may result in pairs or chains of cells Pairs = diplococci
Example: Neisseria gonorrhoeae Chains = streptococci
Example: species of Streptococcus
Morphology of Prokaryotic Cells
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
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
Morphology of Prokaryotic Cells
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
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
The Basic Structural Component of the Membrane: Phospholipid Molecule
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
The Fluid-Mosaic Model of the Membrane Structure
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
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
Cytoplasmic Membrane
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
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
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
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
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
Cytoplasmic Membrane
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
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
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
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
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
TYPICAL PROKARYOTIC CELL
Gram Positive Bacterial Cell Wall
Gram Negative Bacterial Cell Wall
Note thin Peptidoglycan layer inside a Lipopolysaccharide layer
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
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
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 O157:H7 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
Cell Wall
Peptidoglyan (PTG) as a target Many antimicrobial interfere with the synthesis
of PTG Examples include
Penicillin Lysozyme
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
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
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
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
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
Flagella and Pili
Some bacteria have protein appendages Not essential for life
Aid in survival in certain environments They include
Flagella Pili
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
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
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
CHEMOTAXIS
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
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
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
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
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
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
The Schaeffer-Fulton Spore Stain
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
Endospore
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
Eukaryotic Plasma Membrane
Transport across eukaryotic membrane Some molecules pass through membrane via
transport proteins Others taken in through endocytosis and
exocytosis
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
Eukaryotic Plasma Membrane
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
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
Exocytosis Reverse of endocytosis Vesicles inside cell fuse with plasma
membrane Releases contents into external environment
Eukaryotic Plasma Membrane
Protein Structures of Eukaryotic Cell Eukaryotic cells have unique structures that
distinguish them from prokaryotic Cytoskeleton Flagella Cilia 80s ribosome
Protein Structures of Eukaryotic Cell
Cytoskeleton Threadlike proteins Reconstructs to adapt to
cells changing needs Composed of three
elements Microtubules Actin filaments Intermediate fibers
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
Actin filaments Composed of actin polymer Enable cell cytoplasm to move
Assembles and disassembles causing motion Pseudopod formation
Protein Structures of Eukaryotic Cell
Intermediate fibers Function to strengthen cell Enable cells to resist physical stress
Protein Structures of Eukaryotic Cell
Protein Structures of Eukaryotic Cell Flagella
Flexible structure Function in motility 9+2 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
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
Arrangements of Bacterial Flagella
Polar, monotrichous flagellum
Polar, amphitrichous flagellum
Peritrichous flagella
Proteus (29,400X)
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
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
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
Membrane-bound Organellesof Eukaryotes
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