Microbiology

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JerrahAnn
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63454
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Microbiology
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2011-02-02 16:59:36
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  1. Flu and Pnuemonia
    Top 2 Respiratory Disease
  2. Food Microbiology
    • Wine/beer
    • Bread
    • Cheese
    • Yogurt
  3. Bioremediation
    Degrading of harmful chemicals by living organisms

    Example: Pseudomonas in marine oil spills
  4. Natural and Genetically Engineered Bacterial Products
    • Synthesized by bacterial metabolism
    • Ethanol, amino acid
    • Insulin, vaccines, antibiotics by g.e.
  5. Past Medical Microbiology
    • “Conquered” disease: smallpox
    • Production of vaccines and antibiotics
  6. Present and Future Medical Microbiology
    • Model organisms to study
    • Emerging diseases
    • • Swine flu, avian flu, SARS
    • • Changing lifestyles, population expansion, increased global travel
  7. Bacteria
    • Prokaryotes
    • Escherichia coli
    • Single cell
    • Cell walls contain peptidoglycan
  8. Archaea
    • Prokaryotes
    • Pyrolobus fumari
    • Similar in size and shape of bacteria
    • Does not have peptidoglycan
  9. Eucarya
    • Microbial
    • Algae
    • Fungi
    • Protozoa - Giardia
  10. Non-Living Microbes
    • Viruses
    • Viroids
    • Prions
  11. Viruses
    Nucleic acid with a protein coat
  12. Viroids
    RNA causing plant disease
  13. Prions
    Infectious protein causing mad cow disease
  14. Eucaryote Cells
    Prokaryote Cells
    Viruses
    Sizes of cells biggest to smallest
  15. Chemical Components of a Cell
    • Water
    • pH – concentration of H+ moles per liter
    • Organic and inorganic molecules
    • Macromolecules
    • - Proteins
    • - Polysaccharides
    • - Nucleic acids
    • - lipids
  16. Polymers
    Large molecules formed from subunits (monomers)
  17. Synthesis
    Addition of monomers- dehydration
  18. Breakdown
    Withdrawal of water molecule - hydrolysis
  19. Function of Proteins
    • Catalyze reactions
    • Cell movement
    • Uptake of nutrients
    • Gene regulation
    • Regulating cell structure
  20. Amino Acids
    • Each contain a unique side chain, 20 total
    • Grouped according to side chain property
    • - Hydrophilic
    • - Hydrophobic
  21. Peptide Bond
    Linkage of a carboxyl group to an amino group of two amino acids
  22. Primary structure of a Protein
    Single chain of amino acids connected by peptide bonds
  23. Secondary Structure of a Protein
    Arranged according to hydrogen bonds between side chains of amino acids
  24. Tertiary Structure of a Protein
    Some organization; loosely arranged
  25. Quaternary Structure of a Protein
    Includes more than one unit of protein structure held by hydrogen bonds
  26. Hydrophobic
    Fear of water
  27. Hydrophillic
    Love of water
  28. Protein Folding
    Some proteins require assistance by chaperones
  29. Denaturation
    • Protein shape is changed by broken bonds
    • Effected by:
    • • High temperature
    • • High or low pH
    • • Certain solvents
  30. Carbohydrates
    • Carbon (1):Hydrogen (2):Oxygen (1)
    • Monosaccharides
    • Disaccharides
    • Polysaccharides
  31. Monosaccharides
    • Ribose, deoxyribose
    • 5- or 6- carbon sugars found in DNA and RNA
  32. Disaccharides
    Food sources: lactose, sucrose
  33. Polysaccharides
    Part of cell structure: Cellulose, glycogen
  34. Nucleic Acids
    • Nucleotides
    • Genetic Information: DNA, RNA
  35. Nucleotides
    • Carry chemical energy - ATP
    • Part of enzymes – coenzyme A
    • Act as signaling molecule – cyclic AMP
  36. DNA
    • Long, double stranded helix with thymine
    • Contains A, G, T, C
    • Anti Parallel
    • Complementary pairing between:
    • - adenine:thymine
    • - guanine:cytosine
  37. RNA
    • Short, single strand with uracil replacing thymine
    • Contains, A, U, G, C
  38. Bases
    • Purines
    • Pyrimidines
  39. Purines
    Components of RNA and DNA; the two major ones are adenine and guanine
  40. Pyrimidines
    Components of RNA and DNA; the three major ones are thymine, cytosine, and uracil
  41. Joining Nucleotides
    Ester bond between phosphate of 5’ carbon and 3’ carbon
  42. Lipids
    • Major component of membrane
    • Simple Lipids
    • Compound Lipids
  43. Simple Lipids
    • Only carbon, oxygen and hydrogen
    • Fats= fatty acid + glycerol
    • Saturated- no double bonds
    • Unsaturated- double bonds
  44. Compound Lipids
    Phospholipids- a lipid that has a phosphate molecules as part of it's structure
  45. Compound Microscopes
    • Ocular lens magnifies image from objective lens
    • Allows viewing at comfortable distance
  46. Magnification
    • Enlargement of image of a specimen
    • Maximum for light microscopes is 10X for ocular lens & 100X for objective lens = 1000X total
  47. Resolution
    • The ability to distinguish between small objects close together
    • Resolving power of light microscope 200 nm
  48. Contrast
    The number of visible shades in a specimen
  49. Transmission
    Light passes through
  50. Reflection
    • Light bounces off
    • We see the color of an object based on wavelengths of light reflected by its surface
  51. Absorption
    Absorbs light
  52. Fluorescence
    • Light of one wavelength is absorbed
    • Triggers emission of lower energy light of a different wavelength
    • Specimen is stained with fluorescent dye
    • Expose specimen to ultraviolet, violet, or blue light then emits bright blue, orange, green or red
  53. Refraction
    Light bends
  54. Light Microscopy
    • Bright field
    • Phase contrast
    • Dark-field
  55. Bright Field
    • Evenly illuminated field of view
    • Usually requires dye or stain i.e. gram stain, acid fast
  56. Phase Contrast
    Using differences of refracted light
  57. Dark-Field
    • Hollow cone of light is used to illuminate a specimen
    • Object appears bright against a dark background
  58. Basic Dyes & Simple Staining
    • Adhere to negatively charge cell parts
    • Methylene blue, crystal violet, safranin
  59. Acidic Dyes & Negative Staining
    • Stains background; leaves cells colorless
    • Ex: Capsule stain
  60. Differential Stains
    • Gram stain
    • Acid-fast staining
  61. Gram Stain
    Based on membrane structure using crystal violet and safranin
  62. Acid-Fast Staining
    • Stains Mycobacterium
    • Based on high lipid content in membrane
    • Carbol fusion applied over steam bath
  63. Confocal Scanning Laser
    • Specimens stained with flourescent dye
    • Laser scans regions and planes to create 3D image
    • Slices specimen into thin layers
  64. Electron Microscopy
    • Light is replaced by a beam of electrons from tungsten filament in electron gun
    • Sample preparation is complex – viewed in a vacuum
    • Condenser magnet focuses beam on sample
    • Final image focused on fluorescent screen or photographic film
  65. Transmission Electron Microscopy
    • Observe fine cell structure
    • Thin sectioning or freeze fracturing
  66. Scanning Electron Microscopy (SEM)
    Observe surface details
  67. Walking Pnuemonia
    • Leading cause of pneumonia in college students
    • Fever, headache, fatigue, followed with dry cough progressing to production of mucoid sputum
    • Transmitted by aerosolized droplets; symptoms arise after 1 week
  68. Coccus
    • Round
    • Example: Staphylococcus aureus
  69. Bacillus
    • Rod shaped
    • Example: Escherichia coli
  70. Coccobacillus
    • Looks like a bead
    • Example: Haemophilus influenzae
  71. Staphylo-
    Clusters
  72. Vibrio
    • Boomerang shaped
    • Example: Vibrio cholerae
  73. Spirillum
    • Wave shaped
    • Example: Helicobacter pylori
  74. Spirochete
    • Spiral shaped
    • Example: Treponema pallidum
  75. Structure of Cytoplasmic Membrane of Prokaryotic Cell
    • Lipid bilayer embedded with proteins
    • Selectively permeable
    • Hydrophobic interior and hydrophilic exterior
    • Proteins move around lipid layers
    • • Fluid mosaic model
  76. Permeability of Cytoplasmic Membrane
    • Simple Diffusion
    • Facilitated Diffusion
    • Active Transport
  77. Simple Diffusion
    • Small molecules move freely in and out of cell
    • Creates large amount of osmotic pressure on membrane
  78. Facilitated Diffusion
    Carrier protein helps movement from higher to lower concentrations
  79. Active Transport
    Energy used to transport against concentration gradient
  80. Cell Wall of Prokaryotic Cell
    • Required to withstand osmotic pressure from within and prevent cell from bursting
    • - Approx. 75 psi- constantly in aqueous environment with low [solute]
    • Confers cell shape
    • Targeted by penicillin and lysosyme
  81. Cell Wall Structure of Prokaryotic Cell
    • Two groups: gram + and gram -
    • Peptidoglycan
    • - Nacetylmuramic acid (NAM)
    • - N-acetylglucosamine (NAG)
    • Deviants:
    • - Mycoplasma lack a cell wall
    • - Archea do not contain peptidoglycan
  82. Gram Positive Cell Wall of a Prokaryotic Cell
    • Very thick layer of peptidoglycan
    • Contains
    • - Teichoic acid
    • - Often some proteins
    • Retains crystal violet dye of Gram stain even after addition of alcohol
    • Stains purple
  83. Gram Negative Cell Wall of a Prokaryotic Cell
    • Peptidoglycan is much thinner
    • Covered by an outer membrane
    • • Porins, which provide channels for smaller molecules to pass through
    • • High density of lipopolysaccharide
    • Periplasmic space between cell wall & cell membrane
    • Crystal violet stain easily washed off with alcohol
    • Counterstain with safranin
    • Stains red
  84. Flagella
    • Confer swimming motility in liquids
    • Long, rigid, threadlike propellers extending from bacterial surface
  85. Pili
    • Short and thin
    • Used for attachment (fimbriae)
    • - Conjugation
  86. Protoplasm of Prokaryotic Cells
    • Cell pool for all biosynthetic functions
    • Mixture of sugars, amino acids & salts
    • 70-80% water
    • Packed with enzymes
  87. Chromosome of Prokaryotic Cells
    • Irregular mass in nucleoid region
    • Single circular ds DNA
    • Supercoiled
    • Contains essential genes
  88. Plasmids of Prokaryotic Cells
    • Smaller circular ds DNA outside chromosome
    • - Contain genes that enhance survivability but are not necessary
  89. Ribosomes of Prokaryotic Cells
    • Sites of protein synthesis
    • - Thousands per cell
    • Free in cytoplasm or tethered to membrane
    • rRNA + protein
    • Difference in prokaryotic (70S) and eukaryotic (80S) ribosome allows antibiotic targeting
  90. Endospores of Prokaryotic Cells
    • For surviving environmental extremes
    • • Heat, drying, radiation, pH extremes
    • • Conditions common to soil and rock environments, aquatic sediments, mud, deserts-where long periods without water are common
    • All Gram+ to date
    • Most common genera:
    • • Clostridium (tetanus, botulism, gangrene)
    • • Bacillus (anthrax)
    • Have a VERY low water content
  91. Sporulation: Bacterial Differentiation
    • External stimulus
    • Extremes such as heat, dryness, etc.
    • Differentiated gene expression
    • Asymmetrical cell division
  92. Endospore Formers
    • Bacillus anthracis responsible for anthrax scare after 9/11
    • Commonly found in soil
  93. Streptomyces
    • Resemble fungi in form – mycelium
    • Commonly found in soil
    • At tips of filaments are conidia spores
  94. Eukaryotic Cells
    • Size:
    • Cell Wall:
    • Chromosomes:
    • Ribosomes:
    • Organelles:
  95. Prokaryotic Cells
    • Size:
    • Cell Wall:
    • Chromosomes:
    • Ribosomes:
    • Organelles:
  96. Linean System: Two Kingdoms
    Animalia and Plantae
  97. Protista
    • The 3rd kingdom added in the classification in 1866
    • Includes protozoans, algae, fungi, and bacteria
  98. Plantae, Animalia, Fungi, Protista, Prokayotae
    5 Kingdoms Today
  99. Eucarya, archaea, and bacteria
    3 Domains Today
  100. Phenotype and Morphology
    • Size and shape
    • Gram stain
  101. Phenotype and Metabolic Capabilities
    Fermentation of sugars
  102. Phenotype and Serology
    • Identification of bacterial surface proteins or polysaccharides
    • Identify bacterial surface protein or polysaccharide
    • Gram-negative bacteria like E. coli vary in lipopolysaccharide structure
    • • O antigen
    • Flagella can also vary in structure
    • • H antigen
  103. Phenotype and Genomic Typing
    • Pattern of fragment size is restriction fragment length polymorphism (RFLP)
    • Identifying a unique DNA pattern of a particular
    • bacterial strain
    • Using an enzyme that cuts ds DNA at specific
    • points to create several fragments
    • Separate fragments by gel electrophoresis
  104. Microbial Growth
    • Defined as an increase in number
    • Achieved by binary fission
    • Generation or doubling time
    • Nt=N0 x 2n
  105. Biofilms
    • A community of microbes
    • Established by planktonic cells and a slime layer to create a strategic system of cellular signaling and
    • nutrient distribution
    • Examples of location:
    • - Teeth
    • - Sinks
    • - Rocks in streams
  106. Biofilm Problems
    • 65% human infections involve these
    • Treatment of them is difficult
    • Often resistant to antibiotics
    • Causes problems in industry
    • Clogging or damaging pipes or equipment
  107. Biofilm Solutions
    • Extremely helpful inwastewater treatment
    • Helps to breakdown organic products
  108. Pure Culture
    • Taking samples from nature and isolating one
    • species
    • Descended from one cell
    • Sterile instruments, aseptic technique
    • Using streak-plate method, spread culture to single cells
    • Culture medium:
    • - Nutrients in liquid or solid form
    • - Single cell grows into a colony
    • Growth on agar plate
    • - Polysaccharide from marine algae
    • Maintain culture
    • - Restreak cultures regularly
    • - Store long term at -70˚C
  109. Streak Plate Method
    • Pushing bacterial cells across a plate until they are
    • separated individually from each other
    • Individual cells grow into colony of thousands
  110. Cultivating in the Lab
    • Culture Media
    • Complex – unknown content
    • •Meat juices, digested proteins
    • Chemically defined – precise measured chemicals
    • •Salts, sugar, selected growth factors
  111. Types of Culture Media
    • Selective
    • Differential
  112. Selective Culture Media
    • Inhibits growth of some bacteria while allow others to grow
    • MacConkey – inhibits Gram +
  113. Differential Culture Media
    • Distinguishes related organisms based on metabolism or biochemical basis
    • • Blood agar –production of hemolysin
  114. Bacterial Growth in a Lab
    • Closed or batch system
    • - Nutrients are not added
    • - Waste is not removed
    • - Growth curve observed
  115. 1.Lag
    2.Exponential
    3.Stationary
    4.Death
    • Prolonged decline
    4 Stages of Growth
  116. Lag Phase
    • Metabolically active/no increase in number of cells
    • Adaptation; induce enzymes needed
    • Length varies w/ species & conditions
  117. Exponential Phase
    • Population doubles each generation
    • Primary metabolites synthesized
    • Balanced growth- all cellular constituents made at constant rates
    • Most susceptible to antibiotics
  118. Metabolite Production
    • Primary
    • Secondary
  119. Primary Metabolites
    • Amino Acids
    • Nucleic Acids
    • Simple Lipids
  120. Secondary Metabolites
    Antibiotics
  121. Stationary Phase
    • Growth curve horizontal
    • Population growth ceases
    • New cells made at same rate as old cells die (growth rate = death rate)
    • Secondary metabolites are made
  122. Death Phase
    • 99% of population dies
    • Prolonged decline – 1% population mutates according to environment
  123. Exponential, because they’re producing nucleic acid, as well as dividing
    Which growth stage(s) would synthesize the most DNA?
  124. Exponential, because they’re growing as fast as they can
    Lag
    Which growth stage(s) would have the most active ribosomes?
  125. Stationary, because it’s cell wall structure starts to change
    Which growth stage(s) would be the most resistant to antibiotics?
  126. Psychrophiles
    Arctic regions
  127. Psychrotrophs
    Refrigerator
  128. Mesophiles
    Soil, human body
  129. Thermophiles
    Compost heaps
  130. Hyperthermophiles
    Hydrothermal vents
  131. Extreme Thermophiles
    • Archaea
    • Volcanic vents; fissures; hydrothermal vents
    • Pyrolobus fumarii grows between 90° and 113° C in deep sea vents
    • • Makes H2S during metabolism
  132. Obligate Aerobes
    Must have O2
  133. Microaerophiles
    Require small amount of O2
  134. Facultative Anaerobes
    • Grow better if O2 is present
    • • Makes more ATP
    • Can grow without as well
  135. Aerotolerant Anaerobes
    Exclusively ferment; O2 does not harm or help
  136. Obligate Anaerobes
    O2 is toxic
  137. Water Availability and Bacteria Growth
    • Hypertonic environment may cause plasmolysis
    • • Higher concentration of salt
    • Moderate halophiles
    • • Marine enivironment (requires 3% salt)
    • Osmotolerant
    • • Skin surface (as much as 10% salt)- Staphylococcus sp.
    • Extreme halophiles
    • • Dead Sea (requires 9% or more – some archaea
  138. Required Elements for Bacteria Nutrition
    • Autotrophs use CO2 to make carbon products for cell
    • Heterotrophs break down organic carbon to make carbon products
    • • Simple sugars such as glucose
  139. Growth Factors for Bacteria Nutrition
    • Some bacteria make all of their own:
    • • Amino acids, vitamins, purines, pyrimindines
    • Others require them from the environment
    • • Fastidious- require additional factors to grow
  140. Energy Source for Bacteria Nutrition
    • Phototrophs capture energy from sunlight
    • • Cyanobacteria
    • • Purple & green sulfur bacteria
    • Chemotrophs use organic or inorganic molecules for energy
    • - Organic- glucose
    • - Inorganic:
    • • H2 – Hydrogenomonas
    • • H2S; FeS2 – Thiobacillus
    • • NO2 - Nitrobacter
  141. Photoautotroph
    • Energy Source: sunlight
    • Carbon Source: CO2
  142. Photoheterotroph
    • Energy Source: sunlight
    • Carbon Source: organic compounds
  143. Chemolithoautotroph
    • Energy Source: inorganic chemicals (H2, NH3, NO2-, Fe^2+, H2S)
    • Carbon Source: CO2
  144. Chemoorganoheterotroph
    • Energy Source: organic compounds (sugars, amino acids, etc)
    • Carbon Source: organic compounds
  145. Metabolic Pathway Components
    • Enzymes
    • ATP
    • Electron carriers
    • Chemical energy source
    • Precursor metabolites
  146. Metabolic Pathway Components: Enzymes
    • Biological catalysts
    • Active site
    • Substrate complex
    • Specificity
    • “Lock and key”
    • Lower the activation energy
  147. Accessories to Protein Action
    • Cofactors
    • Coenzymes
  148. Cofactors
    Enzymes that need help
  149. Coenzymes
    • Who help enzymes
    • - FAD
    • - NAD+
    • - NADP+
    • - Coenzyme A
  150. Regulation
    • Non-competitive inhibition
    • Competitive inhibition
  151. Non-Competitive Inhibition
    • Allosteric
    • • Feedback inhibition
    • Non-reversible inhibitor damages enzyme
  152. Competitive Inhibition
    • Obstructs substrate
    • Blocking the active site
    • Mimics substrate
    • Sulfa drugs and Folic Acid Synthesis
  153. Allosteric Inhibitor
    Changes active site meaning the substrate no longer fits
  154. Feedback Inhibition
    Excess end product of enzymatic reactions becomes allosteric inhibitor
  155. Metabolic Pathway Components: ATP
    • Immediate donor of free energy
    • Substrate level, oxidative and photo phosphorylation
  156. Metabolic Pathway Components: Electron Carriers
    • Reducing power
    • Lose of electron= oxidized
    • Gain of electron= reduced

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