microscopy and cell structure chapter 3. microscope techniques microscopes microscopes most...

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


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



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



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


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



Phase-Contrast Amplifies differences between refractive indexes of

cells and surrounding medium Uses set of rings and diaphragms to achieve resolution


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


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


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


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



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


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


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


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



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



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



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



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


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


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


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


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


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


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


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



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


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


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


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

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



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

microscopy and cell structure chapter 3. microscope techniques microscopes microscopes most...

Documents