Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

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Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

  1. 1. MICROBIAL GROWTH AY 2012-2013Friday, July 27, 2012
  2. 2. DEFINITION OF MICROBIAL GROWTH • NUMBER OF CELLS • NOT CELL SIZE • e.g. Growing microbes = increase in numbers, accumulating coloniesFriday, July 27, 2012
  3. 3. DEFINITION OF MICROBIAL GROWTH • Note: for coenocytic organisms (multinucleate): growth = increased cell sizeFriday, July 27, 2012
  4. 4. FOR YOU TO GROW....Friday, July 27, 2012
  5. 5. HOW ABOUT THEM?Friday, July 27, 2012
  6. 6. HOW ABOUT THEM?Friday, July 27, 2012
  7. 7. HOW ABOUT THEM?Friday, July 27, 2012
  8. 8. RECALL MICROBIAL NUTRITION CARBON SOURCES Autotrophs CO2 sole or principal biosynthetic carbon source Heterotrophs Reduced, preformed, organic molecules from other organisms ENERGY SOURCES Phototrophs Light Chemotrophs Oxidation of organic or inorganic compounds HYDROGEN AND ELECTRON SOURCES Lithotrophs Reduced inorganic molecules Organotrophs Organic moleculesFriday, July 27, 2012
  9. 9. RECALL MICROBIAL NUTRITION MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE HYDROGEN/ELECTRONS AND MICROORGANISMS CARBON PHOTOLITHOTROPHIC Light energy Algae AUTOTROPHY Inorganic hydrogen/electron Purple and green sulfur donor bacteria CO2 carbon source Blue-green algae (cyanobacteria) PHOTOORGANOTROPHIC Light energy Purple non-sulfur bacteria HETEROTROPHY Organic hydrogen/electron Green non-sulfur bacteria donor Organic carbon source (CO2 may also be used)Friday, July 27, 2012
  10. 10. RECALL MICROBIAL NUTRITION MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE HYDROGEN/ELECTRONS AND MICROORGANISMS CARBON CHEMOLITHOTROPHIC Chemical energy source Sulfur-oxidizing bacteria AUTOTROPHY (inorganic) Hydrogen bacteria Inorganic hydrogen/electron Nitrifying bacteria donor Iron bacteria CO2 carbon source CHEMOORGANOTROPHIC Chemical energy source Protozoa HETEROTROPHY (organic) Fungi Organic hydrogen/electron Most non-photosynthetic donor bacteria Organic carbon sourceFriday, July 27, 2012
  11. 11. REQUIREMENTS FOR MICROBIAL GROWTH •PHYSICAL •CHEMICAL REQUIREMENTS REQUIREMENTS • TEMPERATURE • CARBON • • pH NITROGEN, SULFUR & PHOSPHORUS • OSMOTIC • TRACE ELEMENTS PRESSURE • OXYGEN • ORGANIC GROWTH FACTORSFriday, July 27, 2012
  12. 12. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE • “Most microorganisms grow well at temperatures favored by humans” • 3 primary groups (on the basis of temperature preference) • psychrophiles (cold-loving) • mesophiles (moderate-temperature-loving) • thermophiles (heat-loving)Friday, July 27, 2012
  13. 13. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE MINIMUM, OPTIMUM, MAXIMUMFriday, July 27, 2012
  14. 14. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE • Psychrotrophs: grow between 0°C and 20-30°C; cause food spoilage • Hyperthermophiles : extreme temperatures (members of the archaea)Friday, July 27, 2012
  15. 15. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATUREFriday, July 27, 2012
  16. 16. REQUIREMENTS FOR MICROBIAL GROWTH: pH • RECALL: pH acidity or alkalinity of a solution • acidophiles • neutrophiles • alkaliphilesFriday, July 27, 2012
  17. 17. REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE • Reactions of microorganism in solution based on solute concentration: hypertonic, isotonic, hypotonic • e.g. based on osmotic pressure requirement: Halophiles (obligate/extreme or facultative) • Water activity (aw): water that is available for metabolic processes; i.e. water in food which is not bound to food molecules can support the growth of bacteria, yeasts and molds (fungi) or unbound and available waterFriday, July 27, 2012
  18. 18. REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSUREFriday, July 27, 2012
  19. 19. REQUIREMENTS FOR MICROBIAL GROWTH: CARBON • one of the most important requirements for microbial groth • structural backbone of living matter • e.g. Chemoautotrophs (carbon dioxide) and Chemoheterotrophs (organic materials)Friday, July 27, 2012
  20. 20. REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN • ACCESS: amino acids and proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3– • A few bacteria use N 2 in nitrogen fixationFriday, July 27, 2012
  21. 21. REQUIREMENTS FOR MICROBIAL GROWTH: SULFUR • ACCESS: amino acids, thiamine and biotin • Most bacteria decompose proteins • Some bacteria use SO42– or H2SFriday, July 27, 2012
  22. 22. REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN, SULFUR AND PHOSPHORUS • ACCESS: In DNA, RNA, ATP and membranes • PO is a 4 3– source of phosphorusFriday, July 27, 2012
  23. 23. REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS • iron, copper, molybdenum, zinc • essential for the function of co- factorsFriday, July 27, 2012
  24. 24. REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS • BIOTIN • PYRIDOXINE or VIT B6 • Carboxylation (Leuconostoc) • Transamination (Lactobacillus) • CYANOCOBALAMIN or VIT B12 • NIACIN • Molecular rearrangements (Euglena) • Precursor of NAD and NADP (Brucella) • FOLIC ACID • RIBOFLAVIN or VIT B2 • One-carbon metabolism (Enterococcus) • Precursor of FAD and FMN (Caulobacter) • PANTOTHENIC ACID • THIAMINE or VIT B1 • Fatty acid metabolism (Proteus) • Aldehyde group transfer (BacillusFriday, July 27, 2012
  25. 25. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • “microbes that use molecular oxygen produce more energy from nutrients than microbes that do not use oxygen”Friday, July 27, 2012
  26. 26. Friday, July 27, 2012
  27. 27. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • aerobic bacteria • anaerobic bacteria • microaerophilic bacteriaFriday, July 27, 2012
  28. 28. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • singlet oxygen ( 2 1O -): normal molecular oxygen that has been boosted into a higher- energy state; extremely reactive • hydroxyl radical (OH•): most reactive intermediate form of oxygen formed in cellular cytoplasm by ionizing radiationFriday, July 27, 2012
  29. 29. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • peroxide anion (O22-): toxic; active ingredient in hydrogen peroxide and benzoyl peroxide • SOLUTION: catalase and peroxidaseFriday, July 27, 2012
  30. 30. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • superoxide free radicals (O2-): toxicity is caused by their great instability; they steal an electron from a neighboring molecule, which in turn becomes a free radical, and the cycle continues • SOLUTION: production of superoxide dismutase (SOD): aerobic, FA and aerotolerant anaerobes • convert superoxide free radicals to molecular oxygen and hydrogen peroxideFriday, July 27, 2012
  31. 31. REQUIREMENTS FOR MICROBIAL GROWTH: ORGANIC GROWTH FACTORS • VITAMINS: Unlike humans, most bacteria can synthesize all their own vitamins and are not dependent on outside sources • Some bacteria lack the enzymes needed for the synthesis of certain vitamins, amino acids, purines and pyrimidinesFriday, July 27, 2012
  32. 32. CULTURE MEDIAFriday, July 27, 2012
  33. 33. CULTURE MEDIA • nutrient material prepared for the growth of microorganisms in a laboratory • IMPORTANT TERMS: • inoculum: microbes introduced into a culture medium • culture: microbes that grow and multiply in a culture medium • sterile medium: a pre-requisite = no living microorganismsFriday, July 27, 2012
  34. 34. AGAR • solidifying agent • only a few microbes can degrade it • liquifies at 1000C and solidifies below 400C • pouring temperature: 500C (prevents injury to microbes) • used for the preparation of slants, stabs/deeps, platesFriday, July 27, 2012
  35. 35. TYPES OF CULTURE MEDIA: Chemically-defined Media • exact chemical composition is known • mostly for autotrophic bacteria, fastidious bacteria • Contents: organic growth factors (carbon and energy)Friday, July 27, 2012
  36. 36. TYPES OF CULTURE MEDIA: Complex Media • made up of nutrients including extracts from yeasts, meat or plants, or digests of proteins • exact chemical composition varies from batch to batch • mostly for heterotrophic bacteria and fungiFriday, July 27, 2012
  37. 37. TYPES OF CULTURE MEDIA: Anaerobic Growth Media • “reducing media” • sodium thioglycollate: chemically combine with dissolved oxygen and deplete the oxygen in the culture medium • heated first before use to drive off absorbed oxygenFriday, July 27, 2012
  38. 38. ANAEROBIC CULTURE TECHNIQUESFriday, July 27, 2012
  39. 39. ANAEROBIC CULTURE TECHNIQUESFriday, July 27, 2012
  40. 40. ANAEROBIC CULTURE TECHNIQUESFriday, July 27, 2012
  41. 41. TYPES OF CULTURE MEDIA: Selective & Differential Media • Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation • SELECTIVE: suppress growth of unwanted bacteria and encourage the growth of desired microbesFriday, July 27, 2012
  42. 42. TYPES OF CULTURE MEDIA: Selective & Differential Media •Why it can select: • BSA: Bismuth Sulfite Indicator and Brilliant Green are complementary, inhibiting Gram- positive bacteria and coliforms, allowing Salmonella spp. to grow • SDA: pH 5.6 where fungi can outgrow bacteriaFriday, July 27, 2012
  43. 43. TYPES OF CULTURE MEDIA: Selective & Differential Media • Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation • DIFFERENTIAL: distinguish colonies of desired organisms when grown together with othersFriday, July 27, 2012
  44. 44. TYPES OF CULTURE MEDIA: Differential MediaFriday, July 27, 2012
  45. 45. TYPES OF CULTURE MEDIA: Differential MediaFriday, July 27, 2012
  46. 46. TYPES OF CULTURE MEDIA: Enrichment Media • mostly for soil and fecal samples or when desired microbe is injured • may also be selective • e.g. MRS agar (deMann, Rogosa and Sharpe agar or Lactobacillus agar) • e.g. lactose brothFriday, July 27, 2012
  47. 47. PURE CULTUREFriday, July 27, 2012
  48. 48. PREPARING PURE CULTURE • Julius Richard Petri (1887) • Easy to use, stackable (saving space), requirement for plating methodsFriday, July 27, 2012
  49. 49. OBTAINING PURE CULTURES: Streak PlatingFriday, July 27, 2012
  50. 50. PURE VS MIXED CULTUREFriday, July 27, 2012
  51. 51. CHARACTERIZING COLONIESFriday, July 27, 2012
  52. 52. CULTURE PRESERVATIONFriday, July 27, 2012
  53. 53. WAYS TO PRESERVE YOUR CULTURE •subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationFriday, July 27, 2012
  54. 54. WAYS TO PRESERVE YOUR CULTURE • subculturing •mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationFriday, July 27, 2012
  55. 55. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay •freezing as glycerol stocks • liquid nitrogen storage • lyophilizationFriday, July 27, 2012
  56. 56. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks •liquid nitrogen storage • lyophilizationFriday, July 27, 2012
  57. 57. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage •lyophilizationFriday, July 27, 2012
  58. 58. REVIVAL OF PRESERVED L- DRIED CULTURES http://www.jcm.riken.jpFriday, July 27, 2012
  59. 59. GROWTH OF BACTERIAL CULTURESFriday, July 27, 2012
  60. 60. BACTERIAL DIVISIONFriday, July 27, 2012
  61. 61. OTHER FORMS OF DIVISION BY OTHER MICROBES Budding = Chains of conidiospores Rhodopseudomonas carried externally at the tips of the filaments = Actinomycetes Fragmentation of filaments = ActinomycetesFriday, July 27, 2012
  62. 62. THE MATHEMATICS OF GROWTHFriday, July 27, 2012
  63. 63. CELL DIVISION • Generation time: time required for a microbial population to double • g = mean generation time • g = t/nFriday, July 27, 2012
  64. 64. GENERATION TIME • g = t/nFriday, July 27, 2012
  65. 65. SAMPLE... • Given an initial • Solution: t = 2 density of 4 x 104 • n = [ log (1 x 10 ) – 6 • After 2 hours the log (4 x10 4)]/ cell density became 0.301; n = 4.65 1 x 10 6 • Generation time = • Compute for the (t/n); 2/4.65 or 0.43 generation time hours OR 25.8 minutesFriday, July 27, 2012
  66. 66. GENERATION TIME MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours) Escherichia coli 40 0.35 Bacillus subtilis 40 0.43 Mycobacterium 37 12 tuberculosis Euglena gracilis 25 10.9 Giardia lamblia 37 18 Sacharomyces 30 2 cerevisiaeFriday, July 27, 2012
  67. 67. THE GROWTH CURVEFriday, July 27, 2012
  68. 68. OBTAINING A GROWTH CURVE • The Growth Curve can be obtained via a Batch Culture • Microorganisms are cultivated in a liquid medium and grown as a closed system • Incubated in a closed culture vessel with a single batch of medium and NO fresh medium provided during incubation • SCENARIO: Nutrient concentration decline and concentrations of waste increase during the incubation periodFriday, July 27, 2012
  69. 69. 1. THE LAG PHASE • No immediate increase in cell mass or cell number • Cell is synthesizing new components • Cells retool, replicate their DNA, begin to increase in mass and finally divideFriday, July 27, 2012
  70. 70. 1. THE LAG PHASE • The necessity of a lag phase: • Cells may be old and ATP, essential cofactors and ribosomes depleted • must be synthesized first before growth can begin • Medium maybe different from the one the microorganism was growing previously • new enzymes would be needed to use different nutrients • Microorganisms have been injured and require time to recoverFriday, July 27, 2012
  71. 71. SHORT LAG PHASE • SHORT LAG PHASE (or even absent) • Young, vigorously growing exponential phase culture is transferred to fresh medium of same compositionFriday, July 27, 2012
  72. 72. LONG LAG PHASE • LONG LAG PHASE • Inoculum is from an old culture • Inoculum is from a refrigerated source • Inoculation into a chemically-different mediumFriday, July 27, 2012
  73. 73. 2. THE LOG/ EXPONENTIAL PHASE • Microorganisms are growing and dividing at the maximal rate possible given their genetic potential, nature of medium and conditions under which they are growing • Rate of growth is constant: doubling at regular intervals • The population is most uniform in terms of chemical and physiological properties • Why the curve is smooth: • Because each individual divides at a slightly different momentFriday, July 27, 2012
  74. 74. 3. STATIONARY PHASE • Population growth ceases and the growth curve becomes horizontal (around 109 cells on the average) • Why enter the stationary phase: • Nutrient limitation (slow growth) • Oxygen limitation • Accumulation of toxic waste productsFriday, July 27, 2012
  75. 75. 4. DEATH PHASE • Detrimental environmental changes like nutrient depletion and build up of toxic wastes lead to the decline in the number of viable cells • Usually logarithmic (constant every hour) • DEATH: no growth and reproduction upon transfer to new medium • NOTE: Death rate may decrease after the population has been drastically reduced due to resistant cellsFriday, July 27, 2012
  76. 76. DIRECT MEASUREMENT • Plate counts • Filtration • Most Probable Number (MPN) • Direct Microscopic CountFriday, July 27, 2012
  77. 77. PLATE COUNTSFriday, July 27, 2012
  78. 78. RECALL: HOW TO COMPUTE CFUFriday, July 27, 2012
  79. 79. FILTRATIONFriday, July 27, 2012
  80. 80. MPNFriday, July 27, 2012
  81. 81. DMCFriday, July 27, 2012
  82. 82. INDIRECT MEASUREMENTS: ESTIMATING BACTERIAL NUMBERS • Turbidity: spectrophotometry estimates • Metabolic Activity • e.g. MBRT for Milk = Class 1. Excellent, not decolorized in 8 hours; Class 2. Good, decolorized in less than 8 hours but not less than 6 hours; Class 3. Fair, decolorized in less than 6 hours but not less than 2 hours; Class 4. Poor, decolorized in less than 2 hours • Dry Weight: for filamentous moldsFriday, July 27, 2012
  83. 83. NEXT MEETING: MICROBIAL METABOLISM & PHYSIOLOGYFriday, July 27, 2012

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