Chapter 03 Lecture Outline PDF

Because learning changes everything. ® Chapter 03 Lecture Outline Nester's Microbiology A Human Perspective, Tenth Edition Denise Anderson, Sarah Salm, Mira Beins © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No...

Because learning changes everything. ® Chapter 03 Lecture Outline Nester's Microbiology A Human Perspective, Tenth Edition Denise Anderson, Sarah Salm, Mira Beins © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC.  A Glimpse of History Hans Christian Joachim Gram (1853 to 1938) Danish physician working at morgue in Berlin Worked for Dr. Carl Friedlander Attempting to identify cause of pneumonia Gram was developing methods to stain bacteria With one method, bacteria stained unequally Some retained dye, others did not Revealed two different kinds of bacteria Basis for modern Gram stain Identifies two major groups of bacteria according to cell wall structure and chemistry Gram-positive and Gram-negative © McGraw Hill, LLC 2  Prokaryotic versus Eukaryotic Cells 1 The study of cells has revealed two fundamental types: prokaryotic and eukaryotic. The cells of all bacteria and archaea are prokaryotic The cells of all animals, plants, protozoa, fungi, and algae are eukaryotic Similarities and differences between these two cell types have consequences to human health. Bacterial cell components are targets for antibacterial medications used to treat infectious diseases. By interfering with the function of components unique to bacteria we can selectively kill or inhibit bacteria without harming the patient. © McGraw Hill, LLC 3  Prokaryotic versus Eukaryotic Cells 2 Generally much smaller than most eukaryotic cells Small size gives the cells a high surface-area-to-volume ratio making it easier for them to take in nutrients and excrete waste products. That small size, however, also makes the cells vulnerable to threats, including predators, parasites, and competitors. Prokaryotes have evolved many unique features that increase their chances of survival. Eukaryotic cells are more complex than prokaryotic cells. Larger Many cellular processes take place within membrane-bound compartments Defined by the presence of the nucleus © McGraw Hill, LLC 4  Prokaryotic Cell Structures Surface layers are cell envelope Cytoplasmic membrane Cell wall Capsule (if present) Cytoplasm Nucleoid Location of chromosome Locomotor appendages (if present) © McGraw Hill, LLC 5  Prokaryotic Cells – Figure 3.1 b: © Science Source Access the text alternative for slide images. © McGraw Hill, LLC 6  The Cytoplasmic Membrane – Figure 3.2 Cytoplasmic membrane defines boundary of cell Phospholipid bilayer embedded with proteins Hydrophobic tails face in; hydrophilic tails face out Proteins serve numerous functions Selective gates Sensors of environmental conditions Enzymes Fluid mosaic model: proteins drift about in lipid bilayer Access the text alternative for slide images. © McGraw Hill, LLC 7  The Cytoplasmic Membrane Bacteria and Archaea have same general structure of cytoplasmic membranes Distinctly different phospholipid compositions Lipid tails of Archaea are not fatty acids Connected differently to glycerol © McGraw Hill, LLC 8  Permeability of Cytoplasmic Membrane – Figure 3.3 Cytoplasmic membrane is selectively permeable O2, CO2, N2, small hydrophobic molecules, and water pass freely Some cells facilitate water passage with aquaporins Other molecules must be moved across membrane via transport systems Access the text alternative for slide images. © McGraw Hill, LLC 9  Permeability of Cytoplasmic Membrane Simple Diffusion Movement from high to low concentration until equilibrium is reached Speed of diffusion depends on concentration The greater the difference in concentration on either side of a membrane, the higher the rate of diffusion © McGraw Hill, LLC 10  Osmosis– Figure 3.4 (1) Diffusion of water across a selectively permeable membrane due to unequal solute concentrations Water diffuses from high water concentration (low solute concentration) to low water concentration (high solute concentration) Water flows from hypotonic to hypertonic solution No net water flow between isotonic solutions Access the text alternative for slide images. © McGraw Hill, LLC 11  Osmosis– Figure 3.4 (2) Environment of prokaryotes are typically dilute (hypotonic) relative to cytoplasm Water flows into the cell where cytoplasm is a concentrated solution (hypertonic) Cell wall prevents cell from bursting Access the text alternative for slide images. © McGraw Hill, LLC 12  Cytoplasmic Membrane in Energy Transformation – Figure 3.5 Electron Transport Chain (ETC) embedded in cytoplasmic membrane Uses energy from electrons to move protons out of cell Creates electrochemical gradient across membrane Energy called proton motive force Harvested to drive ATP synthesis and some forms of transport, motility Access the text alternative for slide images. © McGraw Hill, LLC 13  Transport of Small Molecules Across Cytoplasmic Membrane – Figure 3.6 Cells use transport systems to move nutrients and other small molecules across the cytoplasmic membrane Transporters, permeases or carriers Membrane-spanning Highly specific: a single transporter generally moves only one molecule type Access the text alternative for slide images. © McGraw Hill, LLC 14  Efflux Pumps Transporters called efflux pumps move waste products and other toxic substances out of cells. Important medically because bacterial cells use them to remove antimicrobial medications that have entered Allow the bacterium to withstand the effects of the medication. © McGraw Hill, LLC 15  Facilitated Diffusion - Figure 3.7 A form of passive transport Movement down gradient; no energy required Not typically useful in low-nutrient environments Access the text alternative for slide images. © McGraw Hill, LLC 16  Active Transport - Figure 3.7 Requires energy Moves material against concentration gradient Sometimes driven by proton motive force Example is efflux pump Sometimes driven by ATP (ABC transporter) © McGraw Hill, LLC 17  Group Translocation - Figure 3.7 Common in bacteria Chemically alters compound during passage through cytoplasmic membrane Phosphorylation common Often used by bacteria to bring glucose into the cell © McGraw Hill, LLC 18  Protein Secretion – Figure 3.8 Active movement of proteins out of cell Examples: exoenzymes (extracellular enzymes), external structures Polypeptides tagged for secretion via signal sequence of amino acids Access the text alternative for slide images. © McGraw Hill, LLC 19  The Cell Wall of Prokaryotic Cells Cell wall is strong, somewhat rigid structure that prevents cell from bursting Distinguishes two main types of bacteria: Gram-positive Gram-negative © McGraw Hill, LLC 20  Peptidoglycan – Figure 3.9 A layer of peptidoglycan is found in the cell walls of both gram negative and gram positive bacterial Alternating series of subunits form glycan chains N-acetylmuramic acid (NAM) N-acetylglucosamine (NAG) Tetrapeptide chain attach to the NAM and link glycan chains together Direct link in Gram-negative cells Peptide interbridge in Gram-positive cells Access the text alternative for slide images. © McGraw Hill, LLC 21  peptidoglycan structure Lysozyme hydrolyses the glycosidic bonds that link NAM and NAG: lysozyme © McGraw Hill, LLC  peptidoglycan structure inhibition of cell wall biosynthesis by antibiotics Penicillin inhibits formation of crosslinks in peptidogycan wall binds enzyme: transpeptidase (penicillin binding protein) transpeptidase forms tetra- peptide crosslinks between adjacent glycan chains Penicillin becomes covalently linked to the enzyme’s active site - inhibits it, irreversibly © McGraw Hill, LLC  The Gram-Positive Cell Wall – Figure 3.10 Relatively thick peptidoglycan layer Teichoic acids extend above peptidoglycan layer Gel-like material called periplasm lies below peptidoglycan layer c: Egbert Hoiczyk Access the text alternative for slide images. © McGraw Hill, LLC 24  The Gram-Negative Cell Wall – Figure 3.11 Thin peptidoglycan layer Outside is unique outer membrane d: Egbert Hoiczyk Access the text alternative for slide images. © McGraw Hill, LLC 25  The Gram-Negative Cell Wall 1 Outer membrane: outside layer is lipopolysaccharide (LPS) Signals immune system of invasion by Gram-negative bacteria Small levels elicit response to eliminate invader LPS is also called endotoxin Large amounts accumulating in bloodstream can be deadly Includes Lipid A Recognized by immune system Includes O antigen Can be used to identify species or strains © McGraw Hill, LLC 26  The Gram-Negative Cell Wall 2 Outer membrane blocks passage of many molecules including certain antimicrobial medications Small molecules and ions can cross via porins Secretion systems important in pathogenesis Between cytoplasmic membrane and outer membrane is periplasmic space Filled with gel-like periplasm Exported proteins accumulate unless specifically moved across outer membrane Binding proteins of ABC transport systems © McGraw Hill, LLC 27  Antibacterial Substances That Target Peptidoglycan Interference with peptidoglycan can weaken cell wall and allow cell to burst Penicillin interferes with peptidoglycan synthesis Prevents cross-linking of adjacent glycan chains Usually more effective against Gram-positive bacteria than Gram-negative bacteria Outer membrane of Gram-negative cells blocks access Derivatives have been developed that can cross Lysozyme breaks bonds linking glycan chains Enzyme found in tears, saliva, other body fluids Destroys structural integrity of peptidoglycan molecule Usually more effective against Gram-positive bacteria © McGraw Hill, LLC 28  Bacteria That Lack Cell Wall Some bacteria naturally lack a cell wall Mycoplasma species, one of which causes a mild form of pneumonia, are flexible because they lack a rigid cell wall. Neither penicillin nor lysozyme affects these organisms. Mycoplasma and related bacteria can survive without a cell wall because their cytoplasmic membrane contains sterols which make it stronger than that of other bacteria. © McGraw Hill, LLC 29  Cell Walls of Archaea Members of Archaea have variety of cell walls Probably due to wide range of environments Includes extreme environments Archaea less well studied than Bacteria No peptidoglycan, but some have similar molecule pseudopeptidoglycan Many have S-layers that self-assemble Built from sheets of flat protein or glycoprotein subunits © McGraw Hill, LLC 30 © McGraw Hill, LLC Architecture of major classes of prokaryotic cell envelopes containing surface (S) layers About S-layers: S-layers in Archaea: glycoprotein lattices : wall component composed of subunits with pillar-like, hydrophobic trans-membrane domains (a), or lipid- modified glycoprotein subunits (b). Some Archaea have a rigid wall layer (pseudomurein in methanogens) as intermediate layer between plasma membrane and S-layer (c). In Gram-positive bacteria (d) S-layer proteins are bound to rigid peptidoglycan-containing layer via secondary cell wall polymers. In Gram-negative bacteria (e) S-layer closely associated with lipopolysaccharide of outer membrane. Wikipedia.  Capsules and Slime Layers – Figure 3.12 Gel-like layer outside cell wall protects or allows attachment Capsule: distinct, gelatinous Slime layer: diffuse, irregular Most composed of glycocalyx (sugar shell), but some are polypeptides Once attached to a surface, cells can grow as biofilm Polymer-encased community Example: dental plaque Some capsules allow bacteria to evade host immune system a–b: Scimat/Science Source © McGraw Hill, LLC 34 capsules and slime layers Bacteria with capsules attaching to intestinal capsule cells(TEM) Bactria adhering to each other in a layer of slime (SEM) © McGraw Hill, LLC capsules and slime layers allow bacteria to adhere to surfaces glycocalyx: extracellular polymer of glycoprotein (polysaccharide) protective outer layer not all bacteria have one if thick and sturdy, a capsule. if thin and diffuse, a slime layer Capsules, viewed by negative staining. capsule considered virulence factor - enhance ability of pathogenic bacteria to evade phagocytosis attach to surfaces be protected from toxins, detergents, bacteriophages  Flagella – Figure 3.13 Flagella involved in motility Spin like propellers to move cell Some important in disease Helicobacter pylori Numbers and arrangements help with characterization of bacteria Peritrichous – flagella distributed around surface of the cell Polar - a single flagellum at one end a: USDA/Science Source; b: Dennis Kunkel Microscopy/SPL/Science Source © McGraw Hill, LLC 38  Flagella – Figure 3.14 Three parts of bacterial flagellum: Basal body: anchors to cell wall and cytoplasmic membrane Hook Filament: made up of flagellin subunits Archaella (flagella of Archaea): Chemically distinct from those of Bacteria About half the diameter of bacterial flagella Use energy from ATP instead of proton motive force Access the text alternative for slide images. © McGraw Hill, LLC 39 MONOTRICHOUS: single flagellum at one end Caulobacter LOPHOTRICHOUS: crescentus Vibrio cholerae, Pseudomonas aeruginosa, Isiomarina flagella in a tuft at one loihiensis end Vibrio fischeri, Helicobacter pylori SPIROCHETES: specialized flagella PERITRICHOUS: flagella are inside periplasm causes corkscrew distributed © McGraw Hill, LLC all over the cell E. coli, motion Borerelia, Treponema and Leptospira  Chemotaxis – Figure 3.15 Chemotaxis: Bacteria sense a chemical and move toward it (nutrient) or away from it (toxin) Movement is series of runs (straight line) and tumbles (changes in direction) due to coordinated rotation of flagella Access the text alternative for slide images. © McGraw Hill, LLC 41  Chemotaxis – Figure 3.16 Other responses observed: Aerotaxis (respond to O2) Magnetotaxis (respond to earth’s magnetic field) Thermotaxis (respond to temperature) Phototaxis (respond to light) Dennis Kunkel Microscopy/SPL/Science Source © McGraw Hill, LLC 42  Pili – Figure 3.17 Pili (singular: pilus) are shorter and thinner than flagella and the function is different Common pili, or fimbriae, allow the bacterial cells to attach to specific surfaces Some pili help bacterial cells move with a twitching or gliding motility Sex pili are used to join one bacterium to another for DNA transfer a: Source: U.S. Department of Agriculture/Harley W. Moon; b: Dennis Kunkel/SPL/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 43  Internal Components of Prokaryotic Cells – Figure 3.18 Chromosome forms gel-like region: the nucleoid Single circular double-stranded DNA molecule Packed tightly via binding proteins and supercoiling Plasmids Do not encode essential genetic information Similar structure to chromosome, but much smaller May be shared with other bacteria; antibiotic resistance can spread this way CNRI/SPL/Science Source © McGraw Hill, LLC 44  Internal Components of Prokaryotic Cells – Figure 3.19 Ribosomes involved in protein synthesis Facilitate joining of amino acids Relative size and density expressed as S (Svedberg unit) that reflects how fast they settle when centrifuged Prokaryotic ribosomes are 70S Composed of 30S and 50S subunits Eukaryotic ribosomes are 80S Important medically: antibiotics impacting 70S ribosome do not affect 80S ribosome Access the text alternative for slide images. © McGraw Hill, LLC 45  Internal Components of Prokaryotic Cells Cytoskeleton: interior protein framework Bacterial proteins similar to eukaryotic cytoskeleton have been characterized Likely involved in cell division and controlling cell shape Storage granules: accumulations of polymers Synthesized from nutrients available in excess Carbon, energy storage: Glycogen Poly-β-hydroxybutyrate (PHB) Metachromatic granules stain red with methylene blue Protein-based compartments: separate reactions or functions Small rigid structures Identical protein subunits forming the semi-permeable container Functions may be more diverse than previously recognized © McGraw Hill, LLC 46  Protein Based Compartments Small, rigid structures that physically separate certain reactions or functions from the cell's cytosol Gas vesicles - provide aquatic prokaryotes with a mechanism of adjustable buoyancy Allowing cells to move up or down in the water column. Allow only gases to flow in freely, thereby decreasing the density of the cell Bacterial microcompartments (BMCs) - contain enzymes required for certain metabolic reactions. By confining these in a compartment, the cell prevents unwanted side reactions and protects the cytosol from toxic metabolites. Encapsulin nanocompartments - the most recently discovered type of compartment. hold certain proteins in isolation (example an iron binding protein) © McGraw Hill, LLC 47  Endospores – Figure 3.20 Endospores: unique type of dormant cell Produced by members of Bacillus, Clostridium May remain dormant for 100 years or longer Extremely resistant to heat, desiccation, chemicals, ultraviolet light, boiling water Can germinate to become vegetative cells that can multiply Found virtually everywhere Dr. Kari Lounatmaa/Science Source © McGraw Hill, LLC 48  Endospores Sporulation triggered by limited carbon or nitrogen Starvation begins 8-hour process Endospore layers prevent damage Exclude molecules (for example, lysozyme) Cortex maintains core in dehydrated state, protects from heat Core has small proteins that bind to and protect DNA Calcium dipicolinate seems to play important protective role Germination triggered by heat, chemical exposure Not a means of reproduction © McGraw Hill, LLC 49  Endospores – Figure 3.21 Access the text alternative for slide images. © McGraw Hill, LLC 50  Eukaryotic Cell Structure and Their Functions – Figure 3.22a and b Eukaryotic cells are highly variable Protozoa are self-contained units with no cell wall Animal cells also lack a cell wall Fungal cells have a cell wall containing polysaccharides such as chitin Plant cells have a cell wall composed of cellulose Access the text alternative for slide images. © McGraw Hill, LLC 51  Eukaryotic Cell Structures and Their Functions – Figure 3.22c Eukaryotic cells are larger and more complex than prokaryotic cells Membrane-enclosed compartments called organelles Nucleus contains cell’s DNA Cells within an organism vary Similar cells form tissues Various tissues form organs c: ©Dr. Thomas Fritsche Access the text alternative for slide images. © McGraw Hill, LLC 52  Eukaryotic Cell Structures and Their Functions – Figure 3.23 Membrane-bound vesicles bud off from one organelle and fuse with another, delivering material to lumen of organelle Access the text alternative for slide images. © McGraw Hill, LLC 53  Cytoplasmic Membrane of Eukaryotic Cells Similar to that of prokaryotic cells Phospholipid bilayer embedded with proteins Proteins in outer layer serve as receptors Bind specific molecule called a ligand Important in cell communication May contain sterols to increase strength Cholesterol in mammals, ergosterol in fungi Lipid rafts: allow cell to detect, respond to signals Many viruses use to enter, exit cells Electrochemical gradient maintained by sodium or proton pumps © McGraw Hill, LLC 54  Transfer of Molecules Across Cytoplasmic Membrane Aquaporins: water passage Channels: small gated pores, allow small molecules or ions to diffuse Carriers: facilitated diffusion, active transport © McGraw Hill, LLC 55  Transfer of Molecules Across Cytoplasmic Membrane – Figure 3.24 and 3.25 Endocytosis: cell takes up material from surrounding environment by forming invaginations in cytoplasmic membrane Exocytosis: internal vesicles fuse with the cytoplasmic membrane and release their contents Access the text alternative for slide images. © McGraw Hill, LLC 56  Endocytosis Pinocytosis most common in animal cells Forms endosome, which fuses to lysosomes Material degraded in endolysosome Receptor-mediated endocytosis allows cell to take up specific extracellular ligands that bind to surface receptors Phagocytosis used by protozoa, phagocytes to engulf Pseudopods surround, bring material into phagosome Phagosome fuses with lysosome to form phagolysosome where material is degraded © McGraw Hill, LLC 57  Secretion Proteins destined for secretion carry a signal sequence that acts as a tag Ribosomes synthesizing protein with a signal sequence attach to endoplasmic reticulum (ER) Protein enters lumen of ER Membrane of ER buds off and binds to cytoplasmic membrane, releasing protein outside the cell Proteins going to other organelles have specific tags © McGraw Hill, LLC 58  Protein Structures Within Eukaryotic Cells Ribosomes: protein synthesis Eukaryotic ribosome is 80S, made up of 60S and 40S subunits Prokaryotic ribosomes are 70S Antibacterial medications typically do not interfere with 80S ribosome © McGraw Hill, LLC 59  Protein Structures Within Eukaryotic Cells – Figure 3.26 Cytoskeleton: cell framework Actin filaments (microfilaments) allow movement Polymers of actin polymerize and depolymerize Microtubules are thickest component Long, hollow structures made of tubulin Found in mitotic spindles, cilia, flagella Framework for organelle and vesicle movement Intermediate filaments provide mechanical support Access the text alternative for slide images. © McGraw Hill, LLC 60  Perspective 3.1 Pathogens Hijacking Actin Some pathogens control polymerization and depolymerization of host cell’s actin ©Pascale F. Cossart ©Mark A. Jepson, University of Bristol SPL/Science Source © McGraw Hill, LLC 61  Protein Structures Within Eukaryotic Cells – Figure 3.27 Flagella and cilia function in motility Covered by extensions of cytoplasmic membrane Composed of microtubules in 9 + 2 arrangement Eukaryotic flagella are very different from prokaryotic flagella Propel cell or pull it forward Cilia are shorter than flagella, move synchronously Can move cell forward or move material past stationary cells Access the text alternative for slide images. © McGraw Hill, LLC 62  Membrane-Bound Organelles of Eukaryotic Cells – Figure 3.28 Nucleus contains the genetic information Surrounded by two phospholipid bilayer membranes Nuclear pores allow large molecules to pass Nucleolus is region where ribosomal RNAs synthesized b: Biophoto Associates/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 63  Mitochondria – Figure 3.29 Generate ATP Bounded by two phospholipid bilayers Inner membrane forms folds (cristae), increasing surface area for ATP generation Mitochondrial matrix contains DNA, 70S ribosomes b: Keith Porter/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 64  Endosymbiotic Theory Ancestors of mitochondria and chloroplasts were bacteria residing within other cells Endosymbiont lost key features (cell wall, replication) as partners because indispensable to one another Lines of evidence Carry DNA for some ribosomal proteins, ribosomal RNA Ribosomes similar to bacterial 70s ribosomes Double membrane surrounds both organelles Multiplication is by binary fission Mitochondrial DNA sequences resemble those of obligate intracellular parasites: rickettsias Chloroplast DNA sequences resemble those of cyanobacteria © McGraw Hill, LLC 65  Chloroplasts – Figure 3.30 Sites of photosynthesis Found only in plants, algae Harvest sunlight energy to generate ATP ATP used to convert CO2 to sugar and starch Contain DNA and 70S ribosomes, two membranes Stroma contains thylakoids containing pigments that capture radiant energy Dr. Jeremy Burgess/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 66  Endoplasmic Reticulum (ER) – Figure 3.31 System of flattened sheets, sacs, tubes Rough endoplasmic reticulum dotted with ribosomes Synthesize proteins not destined for cytoplasm Smooth endoplasmic reticulum: lipid synthesis and degradation, calcium storage Don W. Fawcett/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 67  Golgi Apparatus – Figure 3.32 Membrane-bound flattened compartments Macromolecules synthesized in ER are modified Addition of carbohydrate, phosphate groups Molecules sorted and delivered in vesicles Biophoto Associates/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 68  Other Membrane-Bound Organelles Lysosomes contain degradative enzymes Could destroy cell if not contained Endosomes, phagosomes fuse with lysosomes Material taken up by cell is degraded Old organelles, vesicles fuse with lysosomes: autophagy Peroxisomes use O2 to degrade lipids, detoxify chemicals Peroxisome generates, contains, and ultimately degrades hydrogen peroxide, superoxide Protects cell from toxic effects of these molecules © McGraw Hill, LLC 69  Microscopes Light microscope can magnify 1,000x Common, important tool in microbiology Electron microscope (1931) can magnify more than 100,000x Scanning probe microscope (1980s) can produce images of individual atoms on a surface © McGraw Hill, LLC 70  Principles of Light Microscopy 1 Light passes through specimen and then series of magnifying lenses Most common type is Bright-field microscope - evenly illuminates the field of view and generates a bright background Three key concepts: Magnification: apparent increase in size Resolution: resolving power, or ability to distinguish two objects that are very close together Contrast: difference in color intensity between an object and the background; determines how easily cells can be seen © McGraw Hill, LLC 71  Principles of Light Microscopy 2 Magnification: apparent increase in size Modern compound microscope has two lens types: objective and ocular Magnification is product of objective (4x, 10x, 40x, or 100x) and ocular lens (10x) Condenser lens (between light source and specimen) focuses light on specimen; does not magnify © McGraw Hill, LLC 72  Principles of Light Microscopy – Figure 3.33 Scenics & Science/Alamy Access the text alternative for slide images. © McGraw Hill, LLC 73  Principles of Light Microscopy 3 Resolution: resolving power, or ability to distinguish two objects that are very close together Defined as minimum distance between two points at which those points can be observed as separate Depends on quality and type of lens, wavelength of light, magnification, and specimen preparation Maximum resolving power of light microscope is 0.2 micrometer © McGraw Hill, LLC 74  Principles of Light Microscopy – Figure 3.34 Immersion oil used to displace air between lens and specimen when using high powered 100x objective Has same refractive index (measure of speed of light passing through medium) as glass Prevents refraction of light; keeps rays from missing opening in objective lens Lisa Burgess/McGraw-Hill Education Access the text alternative for slide images. © McGraw Hill, LLC 75  Principles of Light Microscopy – Figure 3.35 Contrast determines how easily cells can be seen Transparent bacteria lack contrast, difficult to see against colorless background Stains increase contrast but kill microbes Lisa Burgess/McGraw-Hill Education © McGraw Hill, LLC 76  Dark-Field Microscope – Figure 3.36 Cells appear as bright objects against dark background Directs light toward specimen at angle Only light scattered by specimen enters objective lens Lisa Burgess/McGraw-Hill Education © McGraw Hill, LLC 77  Phase-Contrast Microscope – Figure 3.37 Special optics amplify difference between refractive index of dense material and surrounding medium Makes cells and other dense material appear darker Lisa Burgess/McGraw-Hill Education © McGraw Hill, LLC 78  Differential Interference Contrast (DIC) Microscope – Figure 3.38 Like phase-contrast, has special optics that depend upon differences in refractive index Separates light into two beams that pass through specimen and recombine Light waves are out of phase when recombined, yield three- dimensional appearance of image micro_photo/Getty Images © McGraw Hill, LLC 79  Fluorescence Microscope – Figure 3.39 Used to observe cells or materials either naturally fluorescent or tagged with fluorescent dyes Molecules absorb light at one wavelength (usually ultraviolet light) and emit light at longer wavelength Most today are epifluorescent: UV light projected onto, not through, specimen ©Evans Roberts © McGraw Hill, LLC 80  Scanning Laser Microscopes (SLM) – Figure 3.40 Specimens stained with fluorescent dye Fluorescent molecules bind to certain internal compounds; marks their precise location Allows detailed interior views of intact cells May provide 3-dimensional images of thick structures ©A. Harrer, B. Pitts, P. Stewart/MSU-CBE © McGraw Hill, LLC 81  Confocal Microscopy Laser beam illuminates a point on one vertical plane of specimen Mirrors scan laser beam across specimen, illuminating successive planes Each plane represents one fine slice of specimen Computer constructs 3-dimensional image Like a miniature computerized axial tomography (CAT) scan for cells © McGraw Hill, LLC 82  Two-photon Microscopy Similar to confocal, but lower energy used Less damaging to cells; allows time-lapse images Light penetrates deeper to give interior views of relatively thick structures © McGraw Hill, LLC 83  Super-Resolution Microscope – Figure 3.41 Can improve resolution down to 10 nanometers a: ©Dr. Henrik Strah/Newcastle University; b: ©Dr. Henrik Strah/Newcastle University © McGraw Hill, LLC 84  Electron Microscopes – Figure 3.42 Can clearly magnify objects 100,000x Uses electromagnetic lenses, electrons, and a fluorescent screen to replace glass lenses, visible light, and the eye Image photographed as black- and-white electron photomicrograph May be artificially colored Access the text alternative for slide images. © McGraw Hill, LLC 85  Electron Microscopes 1 Wavelength of electrons approximately 1,000 shorter than light Resolving power approximately 1,000-fold greater: approximately 0.3 nanometer © McGraw Hill, LLC 86  Electron Microscopes 2 Lenses and specimen must be in vacuum Air molecules would interfere with electrons Impossible to observe living specimens Results in large, expensive unit and complex specimen preparation Two major types: Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM) © McGraw Hill, LLC 87  Transmission Electron Microscopes – Figure 3.43 Transmission Electron Microscope (TEM): beam of electrons passes through specimen or scatters Depends on density of region: dark areas are dense Thin-sectioning used to view internal details, but process can distort cells Freeze-fracturing, freeze-etching reveal shape of internal structures Newer methods that reduce damage to cells include cryo-electron microscopy (cryo-EM), and cryo-electron tomography for 3-D images a: Lee D. Simon/Science Source; b: Dr. Tony Brain/Science Source © McGraw Hill, LLC 88  Scanning Electron Microscopes – Figure 3.44 Scanning Electron Microscope (SEM): beam of electrons scans over surface of specimen Used to observe surface details Surface coated with thin film of metal Electrons released from specimen are observed Dennis Kunkel Microscopy/SPL/Science Source Yields 3-D effect © McGraw Hill, LLC 89  Scanning Probe Microscopes – Figure 3.45 Detailed images of surfaces Example: atomic force microscope (AFM) Resolving power much greater than that of EM Avoids special preparation required for EM Sharp probe moves across sample’s surface Source: Dr. Mary Ng Mah Lee, National University of Singapore/CDC “Feels” bumps, valleys of atoms Laser measures motion, computer produces surface map © McGraw Hill, LLC 90  Preparing Specimens for Light Microscopy – Figure 3.46 Wet mount uses a drop of liquid specimen overlaid with a coverslip Allows observation of living organisms Can be difficult to see when they are colorless Smear involves drying and fixing specimen before staining to visualize Access the text alternative for slide images. © McGraw Hill, LLC 91  Simple Staining Simple staining uses a single dye to stain the specimen Basic dyes carry positive charge Attracted to negatively charged cellular components Examples are methylene blue and crystal violet Acidic dyes can be used for negative staining Cells repel the negatively charged dye; colorless cells stand out against background Can be done as wet mount © McGraw Hill, LLC 92  Differential Staining Differential staining used to distinguish different groups of bacteria Gram stain most widely used for bacteria Two groups: Gram-positive bacteria and Gram-negative bacteria Reflects fundamental difference in cell wall structure © McGraw Hill, LLC 93  Gram Stain – Figure 3.47 Flood smear with primary stain Rinse and flood with iodine, a mordant that stabilizes the dye in the cell Rinse and briefly add alcohol, a decolorizing agent, to remove dye complex from Gram-negative cells Rinse and flood smear with counterstain that adds a different color to Gram- negative cells Access the text alternative for slide images. © McGraw Hill, LLC 94  Differential Staining – Figure 3.47 Success of Gram stain relies upon length of time of the decolorizing step and age of culture Lisa Burgess/McGraw-Hill Education Access the text alternative for slide images. © McGraw Hill, LLC 95 © McGraw Hill, LLC © McGraw Hill, LLC  Acid-Fast Stain – Figure 3.49 Acid-fast stain used to detect organisms that do not readily take up dyes Detects Mycobacterium species such as causative agents of tuberculosis and Hansen’s disease (leprosy) Cell wall contains high concentrations of mycolic acids Multi-step procedure Primary stain is concentrated red dye Acid-fast cells retain red dye after being flooded with acid-alcohol Methylene blue used as counterstain Source: Dr. George P. Kubica/CDC © McGraw Hill, LLC 98  Capsule Stain – Figure 3.50 Allows observation of gel-like layer that surrounds some microbes Capsules stain poorly, so background is stained to make capsule visible India ink added to wet mount is common method Source: CDC/Dr. Leanor Haley © McGraw Hill, LLC 99  Endospore Stain – Figure 3.51 Allows visualization of endospores, resistant dormant cells often formed by Bacillus and Clostridium Endospore resists Gram stain; appears as clear object Endospore stain uses heat to facilitate uptake of the primary dye malachite green by endospore Counterstain (usually safranin) colors other cells pink Lisa Burgess/McGraw-Hill Education © McGraw Hill, LLC 100  Flagella Stains – Figure 3.52 Uses a substance that makes the dye adhere to thin flagella, making them visible Presence and distribution of flagella can help in identification Peritrichous: flagella surround cell Polar: flagellum on one end Tuft: at one or both ends of cell a: Dr. William A. Clark/CDC; b: Source: Dr. William A. Clark/CDC © McGraw Hill, LLC 101  Fluorescent Dyes and Tags – Figure 3.53 Some dyes bind to structures in all cells Some are changed by cellular processes: can distinguish between living and dead cells a: CDC; b: ©Evans Roberts Immunofluorescence uses fluorescent dye-antibody labels to tag a unique microbe protein © McGraw Hill, LLC 102 Because learning changes everything. ® www.mheducation.com © 2022 McGraw Hill, LLC. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further distribution permitted without the prior written consent of McGraw Hill, LLC.