Download Microscopy and Cell Structure

Microscopy and Cell
Structure
Chapter 3
Microscope Techniques
Microscopes
 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 brightfield 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
Principles of Light Microscopy
 Magnification

Bright field scopes have condenser lens


Has no affect on magnification
Used to focus illumination on specimen
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
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
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
Principles of Light Microscopy
 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
 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
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
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
Microscope Techniques
Dyes 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
Microscope Techniques
Dyes and Staining
 Basic dyes (+) more commonly used than
acidic dyes (-)
 Common basic (+) dyes include

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Methylene blue
Crystal violet
Safrinin
Malachite green
Microscope Techniques
Dyes 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 Techniques
Dyes 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 Techniques
Dyes 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
Microscope Techniques
Dyes 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
Microscope Techniques
Dyes 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 Techniques
Dyes 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
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
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
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
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
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
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
 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
 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
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
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
 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

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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 violetiodine complex of Gram stain
Gram-negative bacterium lose crystal violetiodine 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


If chemical compound is
nutrient


Chemotaxis
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



Extrachromosomal


Generally 0.1% to 10% size of
chromosome
Independently replicating
Encode characteristic

Potentially enhances survival
 Antimicrobial resistance
Internal Structure
 Ribosome


Involved in protein
synthesis
Composed of large and
small subunits


Prokaryotic ribosomal
subunits




Units made of riboprotein
and ribosomal RNA
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



Resistant to damaging conditions


Produced through sporulation
Theoretically remain dormant
for 100 years
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
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
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
Eukaryotic Plasma Membrane
 Exocytosis



Reverse of endocytosis
Vesicles inside cell fuse with plasma
membrane
Releases contents into external environment
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
Protein Structures of
Eukaryotic Cell
 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
 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
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
Polar, monotrichous flagellum
Polar, amphitrichous flagellum
Peritrichous flagella
Proteus (29,400X)
Membrane-bound Organelles
of 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 Organelles
of 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
Membrane-bound Organelles
of 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
Membrane-bound Organelles
of 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