Chapter 3PowerPoint PresentationMicroscopic Technique - the instrumentsPrinciples of light microscopyMagnificationResolution Light rays bend as they pass from air to glass (b/c dif refractive index). Oil and glass have same refractive indexContrastPhotomicrograph of a field of cells of the baker’s yeast Saccharomyces cerevisiae. (a) Bright-fieldSlide 10Fig. 03.08Principles of electron microscopyTypes of electron microscopesTransmission and scanning electron microscopyStains and DyesFig. 03.13Slide 17Fig. 03.15Special stains flagella stainsFluorescent microscopySlide 21Bacterial cellCell morphologyCell groupingSlide 25Prokaryotic cell structureFilamentous protein appendagesFlagellaFlagella structure (Gram negative)Flagella (tumbles and runs)PiliRole of PiliFig. 08.17Transfer of plasmid DNA by conjugation. (a) In this example, the F plasmid of an F+ cell is being transferred to an F- recipient cell. Note the mechanism of rolling circle replication (Figure 9.20 and Figure 16.4).Surface layersglycocalyxRole:Cell wallSlide 39Fig. 03.32Gram-positive cell structureFig. 03.34Table 03.05Some cells lack a cell wall – ex. MycoplasmaCharacteristics of bacteria that lacks cell wallCell boundaryStructure:Role :Selective permeability of the cell membraneRole of membrane proteinProton motive forceFig. 03.26Intracellular StructureFig. 03.431- The chromosomePlasmidsEndosporesSlide 58Spore formationStorage granulesStorage granulesRibosomesSlide 63Flotation of gas vesiculate cyanobacteria from a bloom on a nutrient-rich lake, Lake Mendota, Madison, Wisconsin.Chapter 3•Microscopy and cell structureTopics•Microscopic Techniques - the instruments•Stains•Morphology•Prokaryotic cell structure•Eukaryotic cell structureMicroscopic Technique - the instruments•Light microscopes•Electron microscopesPrinciples of light microscopy•Magnification•Resolution•ContrastMagnificationFigure 3.1Resolution Light rays bend as they pass from air to glass (b/c dif refractive index). Oil and glass have same refractive indexFigure 3.2 Figure 3.3Contrast•Stains provide contrast between bacteria and surrounding media (Refractive index differences)•Phase-contrast microscope•Interference microscope•Dark-field microscope•Fluorescence microscope•Confocal scanning laser microscopePhotomicrograph of a field of cells of the baker’s yeast Saccharomyces cerevisiae. (a) Bright-fieldPhotomicrograph of a field of cells of the baker’s yeast Saccharomyces cerevisiae. (b) Phase contrastDIC differential interference contrast :separate light into 2 beams that pass through the specimen and then recombine. At this stage they are out of phase giving the 3 dimensional appearance of specimenRod shape bacteria tagged with a fluorescent dyeFig. 03.08Principles of electron microscopy•Electrons•Electromagnetic lens•Fluorescent screensTypes of electron microscopes•Transmission- Electrons are transmitted through a specimen- 1-D image•Scanning- Electrons scan the surface of the specimen- 2-D imageTransmission and scanning electron microscopyFigure 3.10 Figure 3.11Stains and Dyes•Differential stains•Special stains•Fluorescent dyesFig. 03.13Fig. 03.15Special stains flagella stainsFigure 3.18endospore stainsFluorescent microscopyBacterial cell• morphology (shape)•Arrangement (grouping)•sizeCell morphologyFigure 3.20Cell grouping Figure 3.22sizeProkaryotic cell structure•Filamentous appendages•Surface layers•Cell wall•Cell membrane•IntracellularFilamentous protein appendages•Flagella•Pili (Fimbriae)FlagellaFigure 3.38Flagella structure (Gram negative)Figure 3.39Flagella (tumbles and runs)Figure 3.40PiliFigure 3.42Role of Pili•Pili (Fimbriae)  numerous and short proteic appendages Role: attachment to specific surfaces–e.g. intestinal lining cell, in pathogenic E. coli.• Sex Pili: longer than fimbriae, less numerous (1 or few). Found only in gram neg. bacteria. •Role –Twitching and gliding –conjugation between cells allowing passage of DNA from 1 cell to another and promoting better adaptation to env. Niches.–cell recognition between bacteria and other microorganisms or bacteria /plants or viruses/ bacteriaFig. 08.17Transfer of plasmid DNA by conjugation. (a) In this example, the F plasmid of an F+ cell is being transferred to an F- recipient cell. Note the mechanism of rolling circle replication (Figure 9.20 and Figure 16.4).Surface layers•Glycocalyx•Cell wall•Cell membraneglycocalyx•composed of polysaccharides•Types of glycocalyx–Capsule: rigid gelatinous layer connected to the cell wall–Slime layer: diffuse, irregular loose aggregates of polysaccharides.Role:•Attachment: form a biofilm that enables attachment on solid surfaces e.g.rocks, pipes, teeth (dental plaque)•Prevent nutrients and water loss•Protection:–from heavy metal toxicity–Host immune systemCell wall•Contains peptidoglycan•Gram-positive•Gram-negative•Role: Rigid structure  determines shape and prevents bursting.•Removed by:–1- Penicillin (and other -lactam drug)–2- Lysozymesbreakdowmn peptidpglycan layerFig. 03.32PeptidoglycanGram-positive cell structureFig. 03.34Gram-negative cell structureTable 03.05Some cells lack a cell wall – ex. MycoplasmaFigure 3.36Characteristics of bacteria that lacks cell wall•(e.g.Mycoplasma spp…)–Pleomorph variable in shape.–Resistance to Penicillin and lysozyme–They have sterols in their cytopl. membrane that give rigidity to cell.Cell boundary•= Cytoplasmic membrane•= Cell membraneStructure:•5 nm thin fluid phospholipid bilayer embedded with proteins•- phospholipid bilayer : hydrophobic center and hydrophilic inner and outer layers.•- Membrane proteinsRole :•1) Selective barrier. Controls what enters and exits the cell. •2) generate of energy (proton motive force) in prokaryotes that respire or photosynthesizeSelective permeability of the cell membrane•Simple diffusion of small hydrophobic molecules O2, CO2 and osmosis of water trhrough the phospholipid bilayer. Does not require Energy •Active Transport of other molecules (amino acids, organic acids and inorganic salts) through membrane proteins  requires Energy (proton motive force, ATP, high energy compound…) in special transport systems using membrane proteinRole of membrane protein–Active transport of nutrients (molecules and ions) that cannot pass through the hydrophobic bilayer into the cell e.g. permease and carriers