B io 120 lecture 3 2012 2013

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B io 120 lecture 3 2012 2013

  1. 1. MICROBIAL CELL BIOLOGY Biology 120 Lecture 3 Reference: Chapter 3 and 4 TORTORATuesday, July 3, 2012
  2. 2. GETTING TO KNOW YOUR BACTERIA • Unit of measure (micrometers or nanometers) • Microscopy = most essential tool in microbial cell biologyTuesday, July 3, 2012
  3. 3. ESSENTIALS IN MICROSCOPY • Simple versus Compound • number of lenses • Brightfield versus Darkfield • background • Light versus Electron Microscopy • light/ beam of electrons • SEM versus TEM • 3D surface/ 2D internal structuresTuesday, July 3, 2012
  4. 4. BRIGHT & DARKTuesday, July 3, 2012
  5. 5. SEM & TEMTuesday, July 3, 2012
  6. 6. WHY OIO?Tuesday, July 3, 2012
  7. 7. PREPARATION OF SPECIMENS FOR MICROSCOPY WET MOUNTTuesday, July 3, 2012
  8. 8. PREPARATION OF SPECIMENS FOR MICROSCOPY WET MOUNTTuesday, July 3, 2012
  9. 9. PREPARATION OF SPECIMENS FOR MICROSCOPY FIXED SMEAR = STAININGTuesday, July 3, 2012
  10. 10. PREPARATION OF SPECIMENS FOR MICROSCOPY • FIXATION • Process by which the internal and external structures of cells and microorganisms are preserved and fixed in position • Inactivates enzymes that might disrupt cell morphology • Toughens cell structures to prevent changes during staining and observation • Usually microbes are killed when fixedTuesday, July 3, 2012
  11. 11. TYPES OF FIXATIONS • preserves overall morphology but not structures within cells • smear ready for staining!!! • Penetrates cells and react with cellular componentsTuesday, July 3, 2012
  12. 12. STAINING •STAINS •salt of + or - ions, one of which is colored (chromophore) •+ basic dye (CV, MB, MG, Sf ) •- acidic dye (Ng) •Types of Staining: SIMPLE, DIFFERENTIAL, SPECIALTuesday, July 3, 2012
  13. 13. POSITIVE AND NEGATIVE STAINING CAN YOU SEE THE DIFFERENCE?Tuesday, July 3, 2012
  14. 14. STAINING • SIMPLE • SPECIAL • e.g. MB, CF, CV, Sf • e.g. negative, endospore, flagella • highlight shapes and staining arrangements • staining special • DIFFERENTIAL structures • e.g. Gram stain and Acid fast stain • differentiate and distinguish one kind of bacteria from anotherTuesday, July 3, 2012
  15. 15. SIMPLE STAININGTuesday, July 3, 2012
  16. 16. GRAM STAININGTuesday, July 3, 2012
  17. 17. GRAM STAININGTuesday, July 3, 2012
  18. 18. ACID FAST STAININGTuesday, July 3, 2012
  19. 19. ACID FAST STAININGTuesday, July 3, 2012
  20. 20. NEGATIVE STAININGTuesday, July 3, 2012
  21. 21. ENDOSPORE STAININGTuesday, July 3, 2012
  22. 22. FLAGELLA STAININGTuesday, July 3, 2012
  23. 23. QUESTIONS?Tuesday, July 3, 2012
  24. 24. RECALL... • PROKARYOTIC ORGANISMS • small, mostly unicellular • bacteria and archaea • DIFFERENCES & VARIATION • bacteria vs archeae • prokaryotes vs eukaryotes (microbes) • FACTORS: morphology, chemical composition, nutritional requirements, biochemical activities, source of energyTuesday, July 3, 2012
  25. 25. THE PROKARYOTESTuesday, July 3, 2012
  26. 26. SIZE, SHAPE & ARRANGEMENT OF BACTERIAL CELLSTuesday, July 3, 2012
  27. 27. WHAT ARE THE 3 BASIC SHAPES OF BACTERIA? •Measure: 0.2-2.0µm (diameter) x 2-8µm (length) •Basic shapes: coccus, bacillus, spiralTuesday, July 3, 2012
  28. 28. COCCI • usually round, can be oval, elongated or flattened on one side • e.g. diplococci (remain in pairs) • e.g. streptococci (remain in chains) • e.g. tetrads (remain attached in cube-like group of 4) • e.g. sarcinae (remain attached in cube-like group of 8) • e.g. staphylococci (divide in multiple planes, grape-like)Tuesday, July 3, 2012
  29. 29. Tuesday, July 3, 2012
  30. 30. Tuesday, July 3, 2012
  31. 31. BACCILI • divide only across their short axis; mostly single rods • e.g. diplobacilli (remain in pairs after division) • e.g streptobacilli (occurs in chains) • e.g. coccobacilli (cocci-like)Tuesday, July 3, 2012
  32. 32. Tuesday, July 3, 2012
  33. 33. Tuesday, July 3, 2012
  34. 34. SPIRALS & CURVED • have on or more twists • never staright • e.g. vibrios (curved rods( • e.g. spirilla (helical, cork-screw, rigid) • e.g spirochetes (helical but flexible)Tuesday, July 3, 2012
  35. 35. Tuesday, July 3, 2012
  36. 36. flagella - rigid axial filament - flexibleTuesday, July 3, 2012
  37. 37. THE OTHERS Stella sp. Haloarcula sp.Tuesday, July 3, 2012
  38. 38. SHAPE = heredity * Monomorphic, maintain a single shape ** Pleomorphic more than one shapeTuesday, July 3, 2012
  39. 39. ORGANIZATION IN A TYPICAL PROKARYOTE STRUCTURE: 1. Structures external to the cell wall 2. the cell wall 3. structures internal to the cell wallTuesday, July 3, 2012
  40. 40. STRUCTURES EXTERNAL TO THE CELL WALLTuesday, July 3, 2012
  41. 41. GLYCOCALYX or SUGAR COAT • secreted on prokaryotic surface • viscous, sticky, gelatinous polymer • composed of polysaccharide, polypeptide or both • made inside the cell and secreted outside • CAPSULE: organized and firmly attached to the cell wall • SLIMY LAYER: unorganized and loosely attached to the cell wallTuesday, July 3, 2012
  42. 42. FUNCTIONS: • contributory to virulence (degree of pathogenicity) • protect pathogen from phagocytosis • attachment to various surfaces for survival • prevent cell from dehydration • viscosity = inhibits movement of nutrients out of the cell • EPS (extracellular polysaccharide) = capsules made up of sugarsTuesday, July 3, 2012
  43. 43. EXAMPLES •Bacillus anthracis (anthrax) •Streptococcus pneumoniae (bacterial pneumonia) •Streptococcus mutans (dental caries)Tuesday, July 3, 2012
  44. 44. OBSERVATION OF GLYCOCALYX •Bacillus anthracis (anthrax) •Streptococcus pneumoniae (bacterial pneumonia) •Streptococcus mutans (dental caries)Tuesday, July 3, 2012
  45. 45. FLAGELLA • long filamentous appendages that propel bacteria • 3 basic parts: • filament = long outermost region (flagellin) • hook = where filament is attached (various protein) • basal body = anchors flagellum to cell wall and plasma membraneTuesday, July 3, 2012
  46. 46. FLAGELLA • atrichous = lacks flagellum • monotrichous = single polar • amphitrichous = tufts at both ends • lophotrichous =two or more on one or both ends • peritrichous = distributed over the entire cellTuesday, July 3, 2012
  47. 47. FLAGELLATuesday, July 3, 2012
  48. 48. DO NUMBERS MATTER?Tuesday, July 3, 2012
  49. 49. DO NUMBERS MATTER?Tuesday, July 3, 2012
  50. 50. DO NUMBERS MATTER?Tuesday, July 3, 2012
  51. 51. DIFFERENCES IN BASAL BODY (Gram + vs Gram -)Tuesday, July 3, 2012
  52. 52. MOTILITY • ability of an organism to MOVE by itself • RUN-TUMBLE-RUN routine • TAXIS: move away or towards a stimuls (chemotaxis, phototaxis)Tuesday, July 3, 2012
  53. 53. FLAGELLA & MOTILITYTuesday, July 3, 2012
  54. 54. FLAGELLAR PROTEINS • H antigen: useful for distinguishing among serovars of bacteria • e.g. E. coli O157:H7 • NOTE: there are at least 50 fifferent H antigens for E. coliTuesday, July 3, 2012
  55. 55. AXIAL FILAMENTS • locomotory structure for spirochetes • also called “endoflagella” • bundles of fibrils that arise at the ends of the cell beneath an outer sheath • spirals around the cellTuesday, July 3, 2012
  56. 56. AXIAL FILAMENTSTuesday, July 3, 2012
  57. 57. FIMBRIAE & PILI • hair-like appendages • shorter, straighter and thinner than flagella • used for attachment (F) and transfer of DNA/conjugation (P) • essential for colonization in Neisseria (F) • NOT for motility!Tuesday, July 3, 2012
  58. 58. FIMBRIAE & PILITuesday, July 3, 2012
  59. 59. THE CELL WALLTuesday, July 3, 2012
  60. 60. CELL WALL • complex, semi-rigid structure • gives the shape of the cell • surrounds the plasma membrane and protects interior from adverse changes in the outside environment • prevents rupture of bacterial cells • contributes to ability of some species to cause disease • site of action of some antibiotics • ALMOST ALL prokaryotes have cell wallsTuesday, July 3, 2012
  61. 61. CELL WALL: Composition & Characteristics • peptidoglycan or murein • N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) • Linked by polypeptidesTuesday, July 3, 2012
  62. 62. CELL WALL: Composition & CharacteristicsTuesday, July 3, 2012
  63. 63. GRAM + CELL WALLTuesday, July 3, 2012
  64. 64. GRAM + CELL WALL Note: lipoteichoic acid provide antigenic variationTuesday, July 3, 2012
  65. 65. GRAM - CELL WALLTuesday, July 3, 2012
  66. 66. GRAM - CELL WALL Note: outer membrane polysaccharides provide antigenic variation; Lipid A is an endotoxinTuesday, July 3, 2012
  67. 67. GRAM + VS GRAM -Tuesday, July 3, 2012
  68. 68. GRAM + VS GRAM -Tuesday, July 3, 2012
  69. 69. ATYPICAL CELL WALL • e.g. Mycoplasma • no walls or have little wall materials • plasma membrane have sterols (help protect from lysis vice the CW)Tuesday, July 3, 2012
  70. 70. ATYPICAL CELL WALL • e.g. Archaea (Halobacterium sp) • may lack walls or have unusual walls composed of polysaccharides and proteins not peptidoglycan (pseudomurein) • PSEUDOMUREIN = lacks the D-amino acids found in bacterial CWs • NOTE: Gram stain not applicableTuesday, July 3, 2012
  71. 71. ATYPICAL CELL WALL • e.g. Mycobacterium and Nocardia • high concentrations of mycolic acids in CWs (60%) • prevents the uptakes of dyes • Note: Gram stain will work only if mycolic acids removedTuesday, July 3, 2012
  72. 72. DAMAGING CELL WALL • antimicrobial drugs (e.g. penicillin) = halts CW synthesis • lysozyme = targets PG backboneTuesday, July 3, 2012
  73. 73. STRUCTURE INTERNAL TO THE CELL WALLTuesday, July 3, 2012
  74. 74. PLASMA MEMBRANE • primarily phospholipids • lacks sterols thus LESS rigidTuesday, July 3, 2012
  75. 75. PLASMA MEMBRANETuesday, July 3, 2012
  76. 76. IMPORTANT STRUCTURES • GLYCOPROTEINS & GLYCOLIPIDS • help protect and lubricate the cell • involved in cell-to-cell interactions (e.g. pathogen binding in inlfuenza)Tuesday, July 3, 2012
  77. 77. CM FUNCTIONS • selective barrier • breakdown of nutrients and production of energy • What happens when CM destroyed? • cell leakageTuesday, July 3, 2012
  78. 78. PHOTOSYNTHETIC STRUCTURES IN THE CM green sulfur bacteria purple non-sulfur bacteria PHOTOSYNTHETIC PIGMENTS IN MEMBRANE FOLDINGS: 1. Chromatophores 2. Chlorosomes 3. Photosynthetic lamellae purple sulfur bacteriaTuesday, July 3, 2012
  79. 79. BACTERIA VS ARCHAEA CM • EUBACTERIA • Ester linkage • Weaker linkage • ARCHAEBACTERIA • Ether linkage • Stronger linkageTuesday, July 3, 2012
  80. 80. READING ASSIGNMENT: MEMBRANE TRANSPORT SYSTEMSTuesday, July 3, 2012
  81. 81. THE NUCLEAR AREA • “NUCLEOID” • contains the bacterial chromosome • not surrounded by a nuclear envelope (membrane) • do not include histonesTuesday, July 3, 2012
  82. 82. THE NUCLEAR AREA • PLASMIDS • extrachromosomal genetic element • replicate independently • Gene: antibiotic resistance, tolerance to toxic metals, toxin production and synthesis of enzymes • can be transferred from one bacterium to another via conjugationTuesday, July 3, 2012
  83. 83. BACTERIAL RIBOSOMES • ALL PROKARYOTES & EUKARYOTES HAVE RIBOSOMES!!! • site of protein synthesis • composed of two units: • protein sub-unit • ribosomal RNA subunit • NOTE: differ from EUK ribosomes in the number of proteins and rRNA molecules they contain and they are less denseTuesday, July 3, 2012
  84. 84. BACTERIAL RIBOSOMES • PROK = 70S ribosomes while EUK = 80S ribosomes • The 70S = 30S (1 rRNA molecule) + 50S (2 rRNA molecules) 16S ribosomal DNA = prokaryotes 18S ribosomal DNA = eukaryotesTuesday, July 3, 2012
  85. 85. BACTERIAL RIBOSOMES • Antimicrobials: • streptomycin and gentamicin = attach to 30S and interfere with protein synthesis • erythromycin and chloramphenicol = attach to 50S and interfere with protein synthesis • THUS only prokaryotes are affected by these antimicrobialsTuesday, July 3, 2012
  86. 86. INCLUSIONS • inclusions = reserve deposits used when supply are deficient • METACHROMATIC GRANULES • POLYSACCHARIDE GRANULES • LIPID INCLUSIONS • SULFUR GRANULES • CARBOXYSOMES • GAS VACUOLES • MAGNETOSOMESTuesday, July 3, 2012
  87. 87. METACHROMATIC GRANULES • “volutin” • large inclusions • stain red with certain blue dyes (e.g. MB) • inorganic phosphate/polyphosphate reserves • used for ATP synthesis • BACTERIA, ALGAE, FUNGI & PROTOZOA • Corynebacterium diphtheriae (diagnostic)Tuesday, July 3, 2012
  88. 88. METACHROMATIC GRANULES “chinese characters” diagnostic for C. diptheriaeTuesday, July 3, 2012
  89. 89. POLYSACCHARIDE GRANULES • consist of glycogen and starch • demonstrated when iodine is applied to cells • appear reddish brown (Glycogen) • appear blue (Starch)Tuesday, July 3, 2012
  90. 90. LIPID INCLUSIONS • Mycobacterium, Bacillus, Azotobacter, Spirillum etc • e.g. poly-B- hydroxybutyric acid (PHBs) • revealed using Sudan dyes (fat soluble dye)Tuesday, July 3, 2012
  91. 91. SULFUR GRANULES • Thiobacillus spp, Beggiatoa Beggiatoa sp. • they derive energy by oxidizing sulfur and sulfur-containing compounds • deposit sulfur granules as energy reservesTuesday, July 3, 2012
  92. 92. CARBOXYSOMES • contain the enzyme 1,5-diphosphate carboxylase (for carbon dioxide fixation) • photosynthetic bacteria, Nitrifying bacteria, Cyanobacteria, ThiobacillusTuesday, July 3, 2012
  93. 93. GAS VACUOLES • hollow cavities in aquatic prokaryotes • cyanobacteria, anoxygenic photosynthetic bacteria and halobacteria • maintain buoyancyTuesday, July 3, 2012
  94. 94. MAGNETOSOMES • inclusion of iron oxide • Magnetospirillum magnetotacticum • used to move downward until they reacha suitable attachment site (act like magnets) • can decompose hydrogen peroxide (to protect cells from its accumulation)Tuesday, July 3, 2012
  95. 95. ENDOSPORES • Clostridium, Bacillus (Bacteria) • Thermoactinomyces vulgaris (Archaea) • specialized resting cells • resistant to adverse conditions (extreme heat, lack of water, exposure to toxic chemicals and radiation) • Dipicolinic acid (DPA) with calcium ions directly involved in spore heat resistanceTuesday, July 3, 2012
  96. 96. SPORULATION/SPOROGENESIS SPORULATION: SPORE FORMATION GERMINATION: SPORE TO VEGETATIVE CELLTuesday, July 3, 2012
  97. 97. ENDOSPORESTuesday, July 3, 2012
  98. 98. THE EUKARYOTESTuesday, July 3, 2012
  99. 99. Tuesday, July 3, 2012
  100. 100. Tuesday, July 3, 2012
  101. 101. EUKARYOTIC FLAGELLA & CILIATuesday, July 3, 2012
  102. 102. For cellular locomotion • Flagella: projections that are few and long in relation to the size of the cell (e.g. Euglena) • Cilia: projections that are numerous and short in relation to the size of the cell (e.g. Tetrahymena) • Difference between prokaryotic flagella: • PROK = rotates • EUK = moves in a wave-like mannerTuesday, July 3, 2012
  103. 103. For cellular locomotionTuesday, July 3, 2012
  104. 104. HOW THEY PROPEL THE CELLTuesday, July 3, 2012
  105. 105. THE 9 + 2 ARRAYTuesday, July 3, 2012
  106. 106. EUKARYOTIC CELL WALL & GLYCOCALYXTuesday, July 3, 2012
  107. 107. CELL WALLS • EUK have simpler cell walls • Algae: cellulose • most Fungi: chitin • Yeasts: glucan and mannan • Protozoa: DO NOT HAVE a typical cell wall = pellicle (flexible outer protein covering)Tuesday, July 3, 2012
  108. 108. GLYCOCALYX • strengthens the cell surface • helps attach cells together • involved in cell to cell recognitionTuesday, July 3, 2012
  109. 109. EUKARYOTIC PLASMA MEMBRANETuesday, July 3, 2012
  110. 110. PLASMA MEMBRANE • similar in function and basic structure with prokaryotes • differences are the proteins found in the membranes • also contain carbohydrates which serves as attachment sites for bacteria and as receptor sites for cell-to-cell recognition • contains sterols (resist lysis due to osmotic pressure)Tuesday, July 3, 2012
  111. 111. PLASMA MEMBRANE • NOTE: group translocation do not occur in eukaryotic membranes • instead ENDOCYTOSIS (e.g. pinocytosis and phagocytosis)Tuesday, July 3, 2012
  112. 112. CYTOPLASM • substance inside the plasma membrane and outside the nucleus • cytosol = fluid portion of the cytoplasmTuesday, July 3, 2012
  113. 113. CYTOPLASM • Major difference: • EUK have complex internal structures (microfilaments, intermediate filaments, microtubules) which forms the cytoskeleton (provides support for cytoplasmic streaming) • many enzymes fund in cytoplasmic fluid of PROK are sequestered in the organelles of EUKTuesday, July 3, 2012
  114. 114. EUKARYOTIC RIBOSOMES & ORGANELLESTuesday, July 3, 2012
  115. 115. RIBOSOMES • same function as in PROK • larger and denser than PROK (80S = 60S with 3 molecules of rRNA; and 40S with 1 molecule of rRNA)Tuesday, July 3, 2012
  116. 116. RIBOSOMES • free ribosomes: unattached, protein synthesis used inside the cell • membrane-bound ribosomes: attached to nuclear membrane and ER, protein synthesis for insertion in the plasma membrane or for export from the cell • polyribosome: located within mitochondria, synthesis of mitochondrial proteins (10-20 ribosomes joined together in a string- like arrangement)Tuesday, July 3, 2012
  117. 117. ORGANELLES • organelle: structure with specific shapes and specialized functions; absent in prokaryotes • Nucleus, ER, golgi complex, lysosomes, vacuoles, mitochondria, chloroplasts, peroxisomes and centrosomesTuesday, July 3, 2012
  118. 118. ORGANELLES • Nucleus = houses the chromosome • ER = transport and storage • Golgi complex = membrane foration and protein secretion • Lysosomes = store digestive enzymes • Vacuoles = storage and rigidityTuesday, July 3, 2012
  119. 119. ORGANELLES • Vacuoles = storage and rigidity • Mitochondria = site of ATP production • Chloroplasts = contain chlorophyll and enzymes for photosynthesis • Peroxisomes = oxidation of organic compiunds (e.g. catalase) destroying hydrogen peroxide) • Centrosomes = contains centrioles for mitotic spindle formationTuesday, July 3, 2012
  120. 120. ORGANELLESTuesday, July 3, 2012
  121. 121. THE EVOLUTION OF EUKARYOTESTuesday, July 3, 2012
  122. 122. HOW THEY CAME ABOUT... • 3.5-4B years ago = simple organisms (similar to prokaryotes) • 2.5B years ago = eukaryotes from prokaryotes • Lyn Margulis: The Endosymbiotic Theory • larger bacterial cells lost their CW and engulfed smaller bacterial cells • endosymbiosis = lives within anotherTuesday, July 3, 2012
  123. 123. ENDOSYMBIOTIC THEORY • ancestral EUK developed a rudimentary nucleus when the plasma membrane folded around the chromosome (NUCLEOPLASM) • Nucleoplasm ingested aerobic bacteria and lived inside it • evolved into a symbiotic relationship (host supply nutrients, while bacteria produce the energy from supplied nutrientsTuesday, July 3, 2012
  124. 124. ENDOSYMBIOTIC THEORY • CHLOROPLASTS = descendants of photosynthetic prokaryotes ingested by the nucleoplasm • FLAGELLA & CILIA = motile spiral bacteria/ spirochetesTuesday, July 3, 2012
  125. 125. NEXT MEETING: JOURNAL REPORTINGTuesday, July 3, 2012

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