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Transcript
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 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 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