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Microbiology Exam 2

Chapters 3, 4, and 5

Zacharias Jansen 1588-1631 The "first" microscope
Hans and Zacharias Jansen 1590's created the first compound microscope
Anthon van Leeuwenhoek Robert Hooke Made improvements by working on the lenses
Anton van Leeuwenhoek First person to observe and describe microorganisms accurately
Magnification Apparent increase in size
Contrast Determines how easily cells can be seen
Resolution Ability of a lens to separate or distinguish small objects that are close together
Shorter wavelength Greater resolution
Refracted Light is refracted (bent) when passing from one medium to another
Refractive Index A measure of how greatly a substance slows the velocity of light
Direction and Magnitude Direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface
Light Properties Absorption and refraction
The Light Microscope Bright-filed microscope Dark-field microscope Phase-contrast microscope Fluorescent microscope
The light Microscope Are compound microscopes Image formed by action of 2 lenses
Bright-Field Microscope Produces a dark image against a brighter background Several objective lenses
Parfocal Microscopes Remain in focus when objectives are changed
Total Magnification Product of the magnifications of the ocular lens and the objective lens
Dark-Field Microscope Produces a bright image of the object against a dark background Used to observe living, unstained preparations
Phase-Contrast Microscope Enhances the contrast between intracellular structures having slight differences in refractive index Excellent way to observe living cells
Phase Contrast Dual beam
Differential Interference Contrast Microscope Creates image by detecting differences in refractive indices and thickness of different parts of specimen Excellent way to observes living cells
Differential Interference Contrast Microscope Like phase-contrast, has special optics that depend upon differences in refractive index. Separates light into 2 beams that pass through specimen and recombine
Differential Interference Contrast Microscope Light waves are out of phase when recombined, yield 3-dimensional appearance of image
DIC Differential Interference Contrast
Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Specimens usually stained with fluorochromes Shows a bright image of the object resulting from the fluorescent light emitted by the speciment
Confocal Scanning Laser Microscope Laser beam used to illuminate spots on specimen Computer compiles images created from each point to generate a 3- dimensional image
Electron Microscopy Beams of electrons are used to produce images Wavelength of electron beam is much shorter than light, resulting in much higher resolution
Transmission Electron Microscope Electrons scatter when they pass through thin sections of a specimen. Transmitted electrons (those that do not scatter) are used to produce image Denser regions in specimen, scatter more electrons and appear darker
Electron Microscopes Electromagnetic lenses, electrons, and fluorescent screen replace glass lenses, visible light, and eye. Image captured on film electron micrograph
Electron Microscopes Wavelength of electrons - 1,000 shorter than light Resolving power- 1,000 fold greater: - 0.2nm
Scanning Electron Microscope Uses electrons reflected from the surface of a specimen to create image Produces a 3-dimensional image of specimen's surface features
Scanning Tunneling Microscope Steady current (tunneling current) maintained between microscope probe and specimen Up and down movement of probe as it maintains current is detected and used to create image of surface of specimen
Atomic Force Microscope (AFM) Sharp probe moves over surface of specimen at constant distance Up and down movement of prove as it maintains constant distance is detected and used to create image
AFM Detailed images of surfaces Resolving power much greater than that of EM Avoid special preparation required for EM
AFM Sharp prove moves across sample's surface Feels bumps, valleys of atoms Laser measures motion, computer produces surface map
Staining Increases visibility of specimen Accentuates specific morphological features Preserves specimens
Fixation Process by which internal and external structures are preserved and fixed in position Process by which organism is killed and firmly attached to microscope slide
Heat Fixing Preserves overall morphology but not internal structures
Chemical Fixing Protects fine cellular substructure and morphology of larger, more delicate organisms
Dyes Make internal and external structures of cell more visible by increasing contrast with background. Have 2 common features
Chromophore Groups Chemical groups with conjugated double bonds Give dye its color Ability to bind cells
Auxochrome Groups Gives the chromophore its acid or base properties
Simple Staining A single staining agent is used Basic dyes are frequently used
Basic dyes Dyes with positive charges Crystal violet, methylene blue
Differential Staining Divides microorganisms into groups based on their staining properties Gram Stain Acid-fast stain
Gram Staining Most widely used differential staining procedure Divides bacteria into two groups based on differences in cell wall structure
Acid-fast Staining Particularly useful for staining members of the genus Mycobacterium High lipid content in cell walls is responsible for their staining characteristics
Negative Staining Often used to visualize capsules surrounding bacteria Capsules are colorless against a stained background
Spore Staining Double staining technique Bacterial endospore is one color and vegetative cell is a different color
Flagella Staining Mordant applied to increase thickness of flagella Flagella commonly used for prokaryotic motility Too thin to be seen with light microscope
Flagella Staining Flagella stain coats flagella to thicken and make visible Presence and distribution can help in identification
Fluorescent Dyes and Tags Some dyes bind to all cells, some only to structures Some are changed by cellular processes: distinguish between living and dead cells
Immunofluorescence Uses antibody to deliver specific fluorescent tag to unique microbe protein
Specimen Preparation Analogous to procedures used for light microscopy For transmission electron microscopy, specimens must be cut very thing Specimens are chemically fixed and stained with electron dense material
Shadowing Coating specimen with a thin film of a heavy metal
Freeze-etching Freeze specimen than fracture along lines of greatest weakness (e.g., membranes)
Similarities between Prokaryotic and Eukaryotic Cells Plasma membrane, DNA and cell wall (plant cells)
Ekuaryotic DNA is in a nucleus surrounded by a nuclear membrane
Prokaryotic DNA is in a nuclear region not surrounded by a membrane
Prokaryotic Cells Have a single circular chromosomes Lack histone proteins Cell wall has peptidoglycan: plant and fungual cells have both cellulose and chitin
Eukaryotic Cells Have paired chromosomes Have histone proteins
Prokaryotic Cell Shapes Coccus, Bacillus (rod), Spirillum
Coccus Spherical
Rod Cylindrical
Pleomorphic Many Shapes
Diplococci Cocci in pairs
Streptococci Cocci in chains
Lactobacillus Rods in chains
Staphylococcus Cocci in clusters
Cell Wall Lies outside the cell membrane in nearly all bacteria
Cell Wall Important Functions Maintains the characteristic shape Prevents the cell from bursting when fluids flow into the cell by osmosis
Components of Bacterial Cell Walls Peptidoglycan (murein): Single most important component Polymer is made up of 2 alternating sugar units 1. N-acetylglucosamine (NAG) 2. N-acetylmuramic acid (NAM
2 Polymers These sugars are joined by short peptide chains that consist of 4 amino acids (tetrapeptides)
Tetrapeptide Chain (string of four amino acids) links glycan chains
Gram Positive Has thick peptidoglycan layer
Teichoic Acid An additional component found in cell walls of gram-positive bacteria. Consists of glycerol, phosphates, and ribitol (sugar alcohol) This polymer extends beyond the rest of the cell wall
Teichoic Acid 2 Functions Attachment site for bacteriophages Passageway for movement of ions in/out of cell
Gram Negative Has thin peptidoglycan layer. Outside is unique outer membrane
Outer Membrane A bilayer membrane found in gram-negative bacteria. Forms the outermost layer of the cell wall; is attached to the peptidoglycan by a continuous layer of lipoprotien molecules
Outer Membrane Proteins called prions form channels through the OM OM has surface antigens and receptors
Lipopolysaccharide An important component of the OM Also called endotoxin; used to ID gram-negative bacteria Released when the cell walls of bacteria are broken down Consists of polysaccharides and Lipid A
Periplasmic Space The area between the cytoplasmic membrane and the plasma membrane in gram-negative bacteria Active area of cell metabolism Contains the cell wall, digestive enzymes and transport proteins Gram positive bacteria lack both an OM and a periplasmic space
Antibacterial Substances That Target Peptidoglycan Peptidoglycan makes good target since unique to bacteria Can weaken to point where unable to prevent cell lysis
Penicillin Interferes with peptidolycan synthesis Prevent cross-linking of adjacent glycan chains Usually more effective against gram-positive bacteria than gra-negative bacteria
Lysozyme Breaks bonds linking glycan chain Enzyme found in tears, saliva, other bodily fluids Destroys structural integrity of peptidoglycan molecule
Acid Fast Bacteria Found in bacteria that belong to the genus, Mycobacterium Cell wall is mainly composed of lipid Lipid component is mycolic acid Acid-fact bacteria stain gram-positive
Gram-Positive Bacteria have a relatively thick layer of peptidoglycan (60-90%)
Gram-Negative Bacteria have a more complex cell wall with a thin layer of peptidoglycan (10-20%)
Acid-Fast Bacteria is thick, like that of gram-positive bacteria, but has much less peptidoglycan and about 60% lipid
Bacteria That Lack a Cell Wall Mycoplasma species have extremely variable shape Penicillin, lysozyme do not affect They are protected from osmotic swelling and bursting by a strengthened cell membrane that contains sterols
Wall Deficient Wall deficient strains are called L-forms
Cell Walls of the Domain Archaea Members of Archaea have variety of cell walls Probably due to wide range of environments Includes extreme environments No peptidoglycan
Domain Archaea Cell Walls Some have similar molecule pseudopeptidoglycan Many have S-layers that self-assemble Built from sheets of flat protein or glycoprotein subunits
Cytoplasmic Membrane Defines boundary of cell Phospholipid bilayer embedded with protiens Hydrophobic tails face in; hydrophilic tails face out Serves as semipermeable membrane
Cytoplasmic Membrane Proteins serve numerous functions: Selective gates Sensors of environmental conditions
Cytoplasmic Membrane Fluid Mosaic model: proteins drift about in lipid bilayer
Internal Cell Structures Cytoplasm Ribosomes Nuclear (Nucleoid) region Vacuoles Certain bacteria sometimes contain endospores
Ribosomes Consist of ribonucleic acid and protein; serve as sits of protein synthesis Abundant in the cytoplasm of bacteria Often grouped in long chains called polyribosomes
Ribosomes 70s in bacteria; 80s in eukaryotes Streptomycin and Erythromycin bind specifically to 70s ribosomes and disrupt bacterial protein synthesis
Nuclear Region (Nucleoid) This centrally located nuclear region consists mainly of DNA, but also contains RNA and protein
DNA Usually one large, circular chromosome
Vibrio cholerae 2 chromosomes, one large and one small
Plasmids Extrachromosomal pieces of smaller, circular
Internal Membrane Systems Photosynthetic bacteria and cyanobacteria contain internal membrane systems Derived from the cell membrane and contain the photosynthetic pigments Nitrifying bacteria also have internal membranes
Inclusions Within the bacterial cytoplasm are a variety of small bodies
Granules Not membrane bound and contain densely compacted substance (glycogen or polyphosphate) Metachromatic Granules (phosphate, volutin) Starch Granules Sulfur Granules
Vesicles Specialized membrane-enclosed structures Lipid Droplets Carboxysomes Gas vesicles
Endospores A specialized resting structure found in bacteria such as Bacillus and Clostridium Helps the bacterial cell survive when conditions become unfavorable
Endospores Highly resistant to heat, drying, acids, bases, certain disinfectants and radiation Calcium and dipicolimic acid
External Structure Many bacteria have structures that extend beyond or surround the cell wall Flagella and pili extend from the cell membrane through the cell wall and beyond Capsules and slime layers surround the cell wall
Monotrichous Bacteria with a single polar flagellum located at one end (pole)
Amphitrichous Bacteria with two flagella, one at each end
Lophotrichous Bacteria with two or more flagella at one or both ends
Peritrichous Bacteria with flagella all over the surface
Atrichous Bacteria without flagella
Chemotaxis Sometimes bacteria move toward or away from substance in their environment by this nonrandom process
Positive chemotaxis Net result is movement towards the attractant (nutrients)
Negative chemotaxis Net result is movement away from the repellent
Cell Structures Axial Filaments
Pili Tiny, hollow projections Used to attach bacteria to surfaces Not involved in movement Long conjugation pilus (sex-pilus) Short attachment pili (fimbriae)
Glycocalyx Capsule Slime layer
Capsule Protective structure outside the cell wall of the organism that secretes it Only certain bacteria are capable of forming capsules
Capsule Chemical composition of each capsule is unique to the strain of bacteria that secreted it Encapsulated bacteria are able to evade hose defense mechanisms (phagocytosis)
Slime Layer Less tightly bound to the cell wall and is usually thinner than a capsule Protects the cell against desiccation (drying out), traps nutrients and binds cells together (biofilm)
Eukaryotic Cells Plasma membrane, cytoplasm, nucleus
Eukaryotic Organelles Mitochondria, chloroplasts, Ribosomes, endoplasmic reticulum, golgi apparatus, vacuoles
Peroxisomes Use O2 to degrade lipids, amino acids, detoxify chemicals
Lysosomes Contain degradative enzymes
Motility Cilia
Motility Pseudopodia
Cell Walls Cellulose-Algae
Cell Walls Chitin-Fungi
Endosymbiont Theory Mitochondria and chloroplasts are appropriate size to be descendants of eubacteria Have inner membranes similar to those on prokaryotic plasma membranes
Endosymbiont Theory Replicate by splitting, as in prokaryotes DNA is circular and different from the DNA of the cell's nucleus Contain their own components for DNA transcription and translation into proteins
Endosymbiont Theory Have ribosomes similar to prokaryotic ribosomes Molecular systematics lend evidence to support this theory Some protists are involved in endosymbiotic relationships
Selectively permeable The membrane allows some things in while keeping other substances out. Polar molecules ions & charged molecules, large molecules = hydrophilic substances- do not cross a lipid bilayer
Passive Transport Cells expends no energy to move substances down a concentration gradient (high to low concentration) Simple Diffusion Facilitated Diffusion Osmosis
Active Transport Cell expends energy, enabling it to transport substances against a concentration gradient
Group Translocation Lower to higher with chemical change Energy from this process is supplied by phosphoenolpyruvate (PEP), a high energy phosphate compound
Bulk Transport endocytosis, phagocytosis, pinocytosis, exocytosis
Osmosis Water movement across a selectively permeable membrane
Facilitated Diffusion
Endocytosis Take up materials via invaginations
Pinocytosis Most common in animal cells Forms endosome, which fuses to lysosomes
Receptor-mediated Endocytosis Is variation Cell internalizes extracellular ligands binding to surface
Phagocytosis Used by protozoa, phagocytes to engulf Pseudopods surround, bring material into phagosome Phagosome fuses with lysosome, phagolysosome
Exocytosis Reverse of endocytosis
Metabolism The sum of all the chemical processes carried out by living organisms The sum of catabolism and anabolism
Anabolism Reactions that require energy to synthesize complex molecules from simpler ones
Catabolism Reactions that release energy by breaking complex molecules into simple ones that can be refused as building blocks
Metabolism All catabolic reactions involve electron transfer which is directly related to oxidation and reduction
Oxidation The loss or removal of electrons
Reduction The gain of electrons
Microorganisms Microorganisms are grouped by energy capture and how they obtain carbon
Autotrophy Use carbon dioxide to synthesize organic molecules
Photoautotrophs Obtain energy from light
Chemoautotrophs Obtain energy from oxidizing simple inorganic substances
Heterotrophy Get their carbon from ready-made organic molecules
Photoheterotrophs Obtain chemical energy from light
Chemoheterotrophs Obtain energy from breaking down ready-made organic compounds
Metabolic Pathway Glycolysis, fermentation, aerobic respiration, and photosynthesis each consist of a series of chemical reaction The product of one reaction serves as the substrate for the next: A>B>C>D
Metabolic Pathway Such chain of reactions is called a metabolic pathway Catabolic pathways capture energy in a form cells can use Anabolic pathways make the complex molecules
Enzymes In general, chemical reactions that release energy can occur without input of energy. The oxidation of glucose releases energy, but the reaction does not occur without an input of energy
Activation Energy The energy required to start such a reaction. Enzymes lower the activation energy so reactions can occur at mild temperatures in living cells
Enzymes Provide a surface on which reactions take place Enzymes generally have a high degree of specificity
Active Site The area on the enzyme surface where the enzyme forms a loose association with the substrate
Substrate The substances on which the enzyme acts
Enzyme-substrate complex Formed when the substrate molecule collides with the active site of its enzyme
Endoenzymes Intracellular
Exoenzymes Extracellular
Active Site and Substrate Each substrate binds to an active site, producing an enzyme substrate complex. The enzyme helps a chemical reaction occur, and one or more products are formed
Coenzymes and Cofactors Many enzymes can catalyze a reaction only if substances called coenzymes, or cofactors are present
Apoenzyme Protein portion of such enzymes
Holoenzyme Nonprotein coenzyme or cofactor that is active when combined with apoenzyme
Coenzyme Nonprotein organic molecule bound to or loosely associated with an enzyme
Cofactor An inorganic ion (e.g. magnesium, zinc) that often improve the fit of an enzyme with its substrate
Energy Transfer by Carrier Molecules Carrier moleules such as cytochromes and some coenzymes carry energy in the form of electrons in many biochemical reactions. Coenzymes such as FAD carry whole hydrogen atoms (electrons together with protons)
Energy Transfer by Carrier Molecules NAD carries one hydrogen atom and one "naked" electron. When coenzymes are reduced, they increase in energy; when they are oxidized, they decrease in energy
Competitive Inhibitor A molecule similar in structure to a substrate can bind to an enzyme's active site and compete with substrate (e.g. sulfa drugs)
Noncompetitive Inhibitors Attach to the enzyme at an allosteric site, which is a site other than the active site. Distort the tertiary protein structure and alter the shape of the active site
Feedback Inhibition Regulates the rate of many metabolic pathways when an end product of a pathway accumulates and binds to and inactivates the first enzyme in the metabolic pathway
Competitive Inhibitor Sulfonamide antibotic, sulfanilamide functions by competitively inhibiting enzyme reactions involving para PABA is needed in enzymatic reactions that produce folic acid, which acts as a coenzyme in the synthesis of purines and pyrimidines
Competitive Inhibitor Mammals do not synthesize their own folic acid so are unaffected by PABA inhibitors, which selectively kill bacteria
Enzymes Most enzymes are proteins
Simple Enzymes Composed of whole proteins
Complex Enzymes Composed of protein plus a relatively small organic molecule
Holoenzyme= apoenzyme + prosthetic group of coenzyme
Factors that Affect Enzyme Reactions Temperature pH Concentration of substrate, product, and enzyme
Temperature and pH Enzymes are affected by heat and extremes of pH The rate at which an enzyme catalyzes a reaction increases with temperature up to the optimum T
Temperature and pH Even small pH changes can alter the electrical charges on various chemical groups in enzyme molecules, thereby altering the enzyme’s ability to bind its substrate and catalyze a reaction
Temperature and pH Most enzymes have an optimum temperature, near normal body temperature, and an optimum pH, near neutral, at which they catalyze a reaction most rapidly
Glycolysis Glycolysis is the metabolic pathway used by most autotrophic and heterotrophic organisms to begin breakdown of glucose. Does not require oxygen, but can occur in presence or absence of oxygen
Phosphorylation The addition of a phosphate group to a molecule, often from ATP and generally increases the molecule's energy
Glycolysis Breaking of a 6 carbon molecule (glucose) into 2 three carbon molecules The transfer of 2 electrons to the coenzyme NAD The capture of energy in ATP
1 glucose = 2 pyruvates Net gain of 2 ATP
Aerobic Metabolism: Respiration Fermentation yields small amount of ATP Partial oxidation of carbon atoms Reduction potential difference between electron donor and acceptor is small (electron tower)
Respiration (aerobic or anaerobic) Substrate molecule's are completely oxidized to CO2 Far higher yield of ATP (aerobic) The Krebs Cycle (aerobic)
Membrane-associated electron carriers Accept and transfer electrons. Conserve energy released for synthesis of ATP
Oxidation/reduction enzymes 1. NADH dehydrogenase 2. Flavoproteins (FAD) 3. Iron-sulfur proteins 4. Cytochromes 5. Quinones (lipid-soluble)
Electron Transport The process leading to the transfer of electrons from substrate to oxygen
Oxidative Phosphorylation Energy releasing dehydrogenation reactions captured in high energy bods as P, combines with ADP to form ATP
The Electron Transport Chain Through a series of oxidation-reduction reactions, the electron transport chain performs 2 basic functions:
The Electron Transport Chain 1. Accepting electrons from an electron donor and transferring them to an electron acceptor 2. Conserving for ATP synthesis some of the energy released during the electron transfer
Chemiosmosis Electrons for the hydrogen atoms removed from the reactions of the Krebs cycle are transferred through the electron transport system Electron transport creates the H potential across the membrane
Chemiosmosis ATP is produced by proton motive force (pmf) by allowing H across the membrane Combination of hydrogen/electron carriers Peter Mitchell 1961
Fermentation Lactic acid fermentation occurs in humans No intermediate; pyruvate accepts electrons from NADH
Fermentation Alcohol fermentation occurs in yeast
Anaerobic Respiration Electron acceptors other than oxygen are used, such as: Inorganic oxygen-containing molecules such as Nitrate (N03-), Sulfate (S042-), Ferric iron (Fe3+), Carbonate (C032-), and Perchlorate (Cl04-)
Anaerobic Respiration Uses only part of the Krebs cycle and electron transport chain Less energy is released Permits microorganisms to respire in anoxic environments
Pentose Phosphate Pathway Alternative to Glycolysis Produces pentoses and NADPH Operates with glycolysis, 1 ATP produced
Entner- Doudoroff Pathway Alternative to glycolysis Produces 2 NADPH and 1 ATP Does not involve glycolysis
Fat Metabolism Most organisms, like most animals, can obtain energy from lipids 1. Fats are hydrolyzed to glycerol and 3 fatty acids 2. Glycerol is metabolized by glycolysis 3. The fatty acids are broken down into 2-carbon pieces by beta-oxidation
Protein Metabolism Proteins can be metabolized for energy They are first hydrolyzed into individual amino acids by proteolytic enzymes Amino acids are deaminated These molecules enter glycolysis, fermentation or the Kreb's cycle
Photosynthesis Plants, algae, several groups of bacteria General reaction is 6CO2 + 12 H2X ----> C6H12O6 + 12X + 6H2O X indicates elements such as oxygen or sulfur
Photosynthesis Light reaction (light-dependent reactions) Capture enrgy and convert it to ATP, NADPH
Photosynthesis Light- independent reactions (dark reactions) Use ATP and NADPH to synthesize organic compounds Involves carbon fixation
Light Reactions Performed by cyanobacteria, algae, and green plants
Carbon Reactions Produce carbohydrates These are the carbon reactions, also known as the Calvin cycle
Photoautotrophy 2 types of photosynthesis in microorganisms: 1. Form similar to plant photosynthesis (evolution of oxygen) Cyanobacteria and algae 2. Bacterial photosynthesis- phototrophic purple sulfur bacteria
Chemoautotrophy (Chemolithotrophs) Energy generation involves inorganic rather than organic chemicals Electron donors are inorganic chemicals such as hydrogen sulfide, hydrogen gas, ferrous iron, and ammonia
Chemoautotrophy (Chemolithotrophs) Aerobic respiration but an inorganic energy source Most chemolithotrophs use carbon dioxide as a carbon source (autotrophs)
Chemolitho(auto)trophs Prokaryotes unique in ability to use reduced inorganic compounds as sources of energy Produced by anaerobic respiration from inorganic molecules serving as terminal electron acceptors Important example of nutrient cycling
Photoheterotrophy Consist of small group of bacteria Purple non-sulfur bacteria, green non-sulfur bacteria
Anabolism: Formation of Macromolecules Two possible sources for monosaccharides, amino acids, fatty acids, nitrogenous bases, and vitamins enter the cell from the outside as nutrients or can be synthesized through various cellular pathways
The Frugality of the Cell Cells have systems for careful management of carbon compounds
Catabolic Pathway Contain strategic molecular intermediates (metabolites) that can be diverted into anabolic pathways A given molecule can serve multiple purposes; maximum benefit can be derived from all nutrients and metabolites of the cell pool
Amphibolism The ability of a system to integrate catabolic and anabolic pathways to improve cell efficiency
Microbial Bioluminescence Bioluminescent bacteria in the petri dish produce enough light to read by. Angler fish lights up the dark, deep-ocean depths with bioluminescent bacteria that live symbiotically in its long “lure” which attracts prey to within reach of its jaws
Created by: rrawls914



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