Download Microscopy and Cell Structure

Microscopy and Cell Structure
Chapter 3
Part I Observing Cells
Microscope Techniques
Microscopes
Principles of Light Microscopy
• Light Microscopy
– Most common and easiest to use: bright-field
microscope
– Important factors in light microscopy include
• Magnification
• Resolution
• Contrast
Principles of Light Microscopy
• Magnification
– two magnifying lenses
• Ocular lens and objective lens
– condenser lens
• focus illumination on specimen
Principles of Light Microscopy
• Resolution
– minimum distance between
two objects that still appear
as separate objects
– determine the usefulness
of microscope
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
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
Principles of Light Microscopy
• Contrast
– Reflects the number of visible shades in a
specimen
– increase contrast
• Use special microscopes
• 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
• Darker appearance for denser materials.
– Uses set of rings and diaphragms to achieve resolution
Principles of Light Microscopy
• Interference Scope
– appear three dimensional
• Depends on differences in
refractive index
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
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
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
Quiz
• With 10x ocular lens and 40x objective
lens, what is the magnifying power?
Quiz
• What are the three important factors for
microscope?
Microscope Techniques
Dyes and Staining
• Dyes and Staining
– stained to observe organisms
– made of organic salts
• Basic dyes carry positive charge
• Acidic dyes carry negative charge
Microscope Techniques
Dyes and Staining
• Common basic dyes include
– Methylene blue
– Crystal violet
– Safrinin
– Malachite green
Microscope Techniques
Dyes and Staining
• Simple staining
– use one color to stain
• increase contrast
between cell and
background
Microscope Techniques
Dyes 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
Microscope Techniques
Dyes and Staining
• Gram Stain
– widely used procedure for classiffying bacteria
– two major groups based on cell wall structural
differences
• Gram positive
• Gram negative
Microscope Techniques
Dyes and Staining
• Gram Stain
– Involves four reagents
• Primary stain
• Mordent
• Decolorizer
• Counter or Secondary stain
Old gram positive appears to be gram negative
Microscope Techniques
Dyes and Staining
• Acid-fast Stain
– Used to stain members of genus
Mycobacterium
• High lipid concentration in cell wall
• Uses heat to facilitate staining
Microscope Techniques
Dyes and Staining
• Acid-fast Stain
– used for presumptive
identification in diagnosis of
clinical specimens
– Requires multiple steps
• Primary dye
• Decolorizer
• Counter stain
Microscope Techniques
Dyes and Staining
• Special Stains
– Capsule stain
– Endospore stain
• Uses heat to facilitate staining
– Flagella stain
Quiz
• What are the two commonly used
differential staining method?
Morphology of Prokaryotic Cells
• Prokaryotes exhibit a
variety of shapes
• Coccus
• Bacillus
– Do not to be confused
with Bacillus genus
Morphology of Prokaryotic Cells
• Coccobacillus
• Vibrio
• Spirillum
• Spirochete
• Pleomorphic
Morphology of Prokaryotic Cells
• groupings morphology
– Cells adhere together after cell division for
characteristic arrangements
• 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
Review of Chapter III part I
Microscope
• Three important factors
• Staining.
• Prokaryotic morphology
Microscopy and Cell Structure
Part II - Prokaryotic Cell Structure
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
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
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
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
Cytoplasmic Membrane
• Simple diffusion• Water, certain gases and small
hydrophobic molecules
• Move along with concentration
gradient
• Osmosis
Cytoplasmic Membrane
• Movement of molecules
across membrane by
transport systems
– Specific
Transport systems include
– Facilitated diffusion
– Active transport
– Group translocation
Directed Movement of Molecules
Across the Cytoplasmic Membrane
Facilitated diffusion
no energy expended
Directed Movement of Molecules
Across the Cytoplasmic Membrane
Facilitated diffusion
Active transport - energy is expended
Moves compounds against a
concentration gradient
Directed Movement of Molecules
Across the Cytoplasmic Membrane
Facilitated diffusion
Active transport - energy is expended
Major facilitator superfamily
ABC transport systems
(expends proton motive force)
(expends ATP)
Example: efflux pumps used in
antimicrobial resistance
Use binding proteins to scavenge and
deliver molecules to transport complex
Example: maltose transport
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
Proton motive force is used to fuel:
•Synthesis of ATP (the cell’s energy currency)
•Rotation of flagella (motility)
•One form of transport
Directed Movement of Molecules
Across the Cytoplasmic Membrane
Facilitated diffusion
Active transport
Group translocation - Chemically modifies a compound during
transport
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)
Prokaryotic structure
• Cell membrane structure
• Movements across membrane
Cell Wall
Provides rigidity to the cell
(prevents it from bursting)
Cell Wall
• Bacterial cell wall
– Rigid structure
– Determines shape of bacteria
– Protection
– Unique chemical structure
• Distinguishes Gram positive from Gram-negative
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
Cell Wall
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 negative—direct join
Gram positive—peptide
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)
Cell Wall
Gram-positive
Thick layer of peptidoglycan
Teichoic acids
Cell Wall
Gram-negative
Thin layer of peptidoglycan
Outer membrane - additional
membrane barrier; porins permit passage
lipopolysaccharide (LPS)
Cell Wall
Gram-negative
Thin layer of peptidoglycan
Outer membrane - additional
membrane barrier; porins permit passage
lipopolysaccharide (LPS)
- ex. E. coli O157:H7
endotoxin
- recognized by innate immune system
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
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
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
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
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
• propeller movements
• more than 100,000
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
– Chemotaxis
• attractant, repellent
• Tumble, run
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)
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
Internal Structures
Genome
Internal Structures
• Some are essential for life
– Chromosome
– Ribosome
• Others 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
• Potentially enhances survival
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
Internal Structures
• Storage granules
– Accumulation of polymers
• Synthesized from excess
nutrient
– Example = glycogen
granules
• Gas vesicles
– Small protein compartments
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
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)
Microscopy and Cell Structure
Part III - Eukaryotic Cell Structure
A BRIEF overview
Membrane-bound organelles
Animal cell
Plant cell
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
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
Eukaryotic Plasma Membrane
• Endocytosis
– Process by which
eukaryotic cells bring in
material from
surrounding
environment
• Pinocytosis
• Phagocytosis
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
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
• 40s +60s
Protein Structures of
Eukaryotic Cell
• Cytoskeleton
– Threadlike proteins
– Reconstructs to adapt
to cells changing
needs
– Composed of three
elements
• Microtubules
• Actin filaments
• Intermediate fibers
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
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
Membrane-bound Organelles
of Eukaryotes
• Nucleus
– Distinguishing feature of
eukaryotic cell
– Two lipid bilayers
– Contains chromosomal
DNA (linear)
– Area of DNA replication
Membrane-bound Organelles
of Eukaryotes
• Endoplasmic
reticulum
– Divided into rough and
smooth
• Rough ER
• Smooth ER
Membrane-bound Organelles
of Eukaryotes
• Golgi apparatus
– a series of membrane bound
flattened sacs
– Modifies macromolecules
produced in endoplasmic
reticulum
• Lysosomes
• Peroxisomes