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B io 120 lecture 3 2012 2013

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  • 1. MICROBIAL CELL BIOLOGY Biology 120 Lecture 3 Reference: Chapter 3 and 4 TORTORATuesday, July 3, 2012
  • 2. GETTING TO KNOW YOUR BACTERIA • Unit of measure (micrometers or nanometers) • Microscopy = most essential tool in microbial cell biologyTuesday, July 3, 2012
  • 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. BRIGHT & DARKTuesday, July 3, 2012
  • 5. SEM & TEMTuesday, July 3, 2012
  • 6. WHY OIO?Tuesday, July 3, 2012
  • 7. PREPARATION OF SPECIMENS FOR MICROSCOPY WET MOUNTTuesday, July 3, 2012
  • 8. PREPARATION OF SPECIMENS FOR MICROSCOPY WET MOUNTTuesday, July 3, 2012
  • 9. PREPARATION OF SPECIMENS FOR MICROSCOPY FIXED SMEAR = STAININGTuesday, July 3, 2012
  • 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. 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. 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. POSITIVE AND NEGATIVE STAINING CAN YOU SEE THE DIFFERENCE?Tuesday, July 3, 2012
  • 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. SIMPLE STAININGTuesday, July 3, 2012
  • 16. GRAM STAININGTuesday, July 3, 2012
  • 17. GRAM STAININGTuesday, July 3, 2012
  • 18. ACID FAST STAININGTuesday, July 3, 2012
  • 19. ACID FAST STAININGTuesday, July 3, 2012
  • 20. NEGATIVE STAININGTuesday, July 3, 2012
  • 21. ENDOSPORE STAININGTuesday, July 3, 2012
  • 22. FLAGELLA STAININGTuesday, July 3, 2012
  • 23. QUESTIONS?Tuesday, July 3, 2012
  • 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. THE PROKARYOTESTuesday, July 3, 2012
  • 26. SIZE, SHAPE & ARRANGEMENT OF BACTERIAL CELLSTuesday, July 3, 2012
  • 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. 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. Tuesday, July 3, 2012
  • 30. Tuesday, July 3, 2012
  • 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. Tuesday, July 3, 2012
  • 33. Tuesday, July 3, 2012
  • 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. Tuesday, July 3, 2012
  • 36. flagella - rigid axial filament - flexibleTuesday, July 3, 2012
  • 37. THE OTHERS Stella sp. Haloarcula sp.Tuesday, July 3, 2012
  • 38. SHAPE = heredity * Monomorphic, maintain a single shape ** Pleomorphic more than one shapeTuesday, July 3, 2012
  • 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. STRUCTURES EXTERNAL TO THE CELL WALLTuesday, July 3, 2012
  • 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. 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. EXAMPLES •Bacillus anthracis (anthrax) •Streptococcus pneumoniae (bacterial pneumonia) •Streptococcus mutans (dental caries)Tuesday, July 3, 2012
  • 44. OBSERVATION OF GLYCOCALYX •Bacillus anthracis (anthrax) •Streptococcus pneumoniae (bacterial pneumonia) •Streptococcus mutans (dental caries)Tuesday, July 3, 2012
  • 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. 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. FLAGELLATuesday, July 3, 2012
  • 48. DO NUMBERS MATTER?Tuesday, July 3, 2012
  • 49. DO NUMBERS MATTER?Tuesday, July 3, 2012
  • 50. DO NUMBERS MATTER?Tuesday, July 3, 2012
  • 51. DIFFERENCES IN BASAL BODY (Gram + vs Gram -)Tuesday, July 3, 2012
  • 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. FLAGELLA & MOTILITYTuesday, July 3, 2012
  • 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. 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. AXIAL FILAMENTSTuesday, July 3, 2012
  • 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. FIMBRIAE & PILITuesday, July 3, 2012
  • 59. THE CELL WALLTuesday, July 3, 2012
  • 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. CELL WALL: Composition & Characteristics • peptidoglycan or murein • N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) • Linked by polypeptidesTuesday, July 3, 2012
  • 62. CELL WALL: Composition & CharacteristicsTuesday, July 3, 2012
  • 63. GRAM + CELL WALLTuesday, July 3, 2012
  • 64. GRAM + CELL WALL Note: lipoteichoic acid provide antigenic variationTuesday, July 3, 2012
  • 65. GRAM - CELL WALLTuesday, July 3, 2012
  • 66. GRAM - CELL WALL Note: outer membrane polysaccharides provide antigenic variation; Lipid A is an endotoxinTuesday, July 3, 2012
  • 67. GRAM + VS GRAM -Tuesday, July 3, 2012
  • 68. GRAM + VS GRAM -Tuesday, July 3, 2012
  • 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. 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. 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. DAMAGING CELL WALL • antimicrobial drugs (e.g. penicillin) = halts CW synthesis • lysozyme = targets PG backboneTuesday, July 3, 2012
  • 73. STRUCTURE INTERNAL TO THE CELL WALLTuesday, July 3, 2012
  • 74. PLASMA MEMBRANE • primarily phospholipids • lacks sterols thus LESS rigidTuesday, July 3, 2012
  • 75. PLASMA MEMBRANETuesday, July 3, 2012
  • 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. CM FUNCTIONS • selective barrier • breakdown of nutrients and production of energy • What happens when CM destroyed? • cell leakageTuesday, July 3, 2012
  • 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. BACTERIA VS ARCHAEA CM • EUBACTERIA • Ester linkage • Weaker linkage • ARCHAEBACTERIA • Ether linkage • Stronger linkageTuesday, July 3, 2012
  • 80. READING ASSIGNMENT: MEMBRANE TRANSPORT SYSTEMSTuesday, July 3, 2012
  • 81. THE NUCLEAR AREA • “NUCLEOID” • contains the bacterial chromosome • not surrounded by a nuclear envelope (membrane) • do not include histonesTuesday, July 3, 2012
  • 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. 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. 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. 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. 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. 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. METACHROMATIC GRANULES “chinese characters” diagnostic for C. diptheriaeTuesday, July 3, 2012
  • 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. 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. 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. CARBOXYSOMES • contain the enzyme 1,5-diphosphate carboxylase (for carbon dioxide fixation) • photosynthetic bacteria, Nitrifying bacteria, Cyanobacteria, ThiobacillusTuesday, July 3, 2012
  • 93. GAS VACUOLES • hollow cavities in aquatic prokaryotes • cyanobacteria, anoxygenic photosynthetic bacteria and halobacteria • maintain buoyancyTuesday, July 3, 2012
  • 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. 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. SPORULATION/SPOROGENESIS SPORULATION: SPORE FORMATION GERMINATION: SPORE TO VEGETATIVE CELLTuesday, July 3, 2012
  • 97. ENDOSPORESTuesday, July 3, 2012
  • 98. THE EUKARYOTESTuesday, July 3, 2012
  • 99. Tuesday, July 3, 2012
  • 100. Tuesday, July 3, 2012
  • 101. EUKARYOTIC FLAGELLA & CILIATuesday, July 3, 2012
  • 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. For cellular locomotionTuesday, July 3, 2012
  • 104. HOW THEY PROPEL THE CELLTuesday, July 3, 2012
  • 105. THE 9 + 2 ARRAYTuesday, July 3, 2012
  • 106. EUKARYOTIC CELL WALL & GLYCOCALYXTuesday, July 3, 2012
  • 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. GLYCOCALYX • strengthens the cell surface • helps attach cells together • involved in cell to cell recognitionTuesday, July 3, 2012
  • 109. EUKARYOTIC PLASMA MEMBRANETuesday, July 3, 2012
  • 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. PLASMA MEMBRANE • NOTE: group translocation do not occur in eukaryotic membranes • instead ENDOCYTOSIS (e.g. pinocytosis and phagocytosis)Tuesday, July 3, 2012
  • 112. CYTOPLASM • substance inside the plasma membrane and outside the nucleus • cytosol = fluid portion of the cytoplasmTuesday, July 3, 2012
  • 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. EUKARYOTIC RIBOSOMES & ORGANELLESTuesday, July 3, 2012
  • 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. 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. 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. 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. 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. ORGANELLESTuesday, July 3, 2012
  • 121. THE EVOLUTION OF EUKARYOTESTuesday, July 3, 2012
  • 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. 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. ENDOSYMBIOTIC THEORY • CHLOROPLASTS = descendants of photosynthetic prokaryotes ingested by the nucleoplasm • FLAGELLA & CILIA = motile spiral bacteria/ spirochetesTuesday, July 3, 2012
  • 125. NEXT MEETING: JOURNAL REPORTINGTuesday, July 3, 2012

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