Biology 120 lecture 4 2011 2012

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Biology 120 lecture 4 2011 2012

  1. 1. REPRODUCTION & GROWTH Lecture 4 Reference: Chapter 6 (Tortora) Parungao-Balolong 2011Thursday, July 14, 2011
  2. 2. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Measurement of Microbial Growth Culture Media Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  3. 3. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Measurement of Microbial Growth Culture Media Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  4. 4. REPRODUCTION IN PROKARYOTES  Binary fission  Budding  Conidiospores (actinomycetes)  Fragmentation of filaments Parungao-Balolong 2011Thursday, July 14, 2011
  5. 5. MICROBIAL GROWTH  Microbial growth = increase in number of cells, not cell size  Nutrients = substances used in biosynthesis and energy production (required for microbial growth)  Environmental Factors = temperature, oxygen levels, osmotic concentration Parungao-Balolong 2011Thursday, July 14, 2011
  6. 6.  GROWTH GROWTH ◦Increase in cellular constituents ◦Leads to a rise in cell number  Budding, Binary Fission  For coenocytic organisms (multinucleate) ◦Growth results in increased cell size not number Parungao-Balolong 2011Thursday, July 14, 2011
  7. 7. MICROBIAL NUTRITION  Macroelements or Macronutrients ◦Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium and iron  Trace elements or Micronutrients ◦Manganese, zinc, cobalt, molybdenum, nickel and copper Parungao-Balolong 2011Thursday, July 14, 2011
  8. 8. GROWTH FACTORS  BIOTIN  PYRIDOXINE or VIT B6 ◦Carboxylation (Leuconostoc) ◦Transamination (Lactobaci!us)  CYANOCOBALAMIN or VIT  NIACIN B12 ◦Precursor of NAD and NADP ◦Molecular rearrangements (Bruce!a) (Euglena)  RIBOFLAVIN or VIT B2  FOLIC ACID ◦Precursor of FAD and FMN ◦One-carbon metabolism (Caulobacter) (Enterococcus)  THIAMINE or VIT B1  PANTOTHENIC ACID ◦Aldehyde group transfer (Baci!us ◦Fatty acid metabolism (Proteus) anthracis) Parungao-Balolong 2011Thursday, July 14, 2011
  9. 9. 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 molecules Parungao-Balolong 2011Thursday, July 14, 2011
  10. 10. 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) Parungao-Balolong 2011Thursday, July 14, 2011
  11. 11. 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 source Parungao-Balolong 2011Thursday, July 14, 2011
  12. 12. THE GROWTH CURVE  Population growth is studied by analyzing the growth curve of microorganisms  Growth of microorganisms reproducing by binary fission can be plotted as the logarithm of cell number versus the incubation time (Growth curve) Parungao-Balolong 2011Thursday, July 14, 2011
  13. 13. THE GROWTH CURVE  The Growth Curve can be obtained via a Batch Culture ◦Microorganisms are cultivated in a liquid medium ◦Grown as a closed system ◦Incubated in a closed culture vessel with a single batch of medium ◦No fresh medium provided during incubation ◦Nutrient concentration decline and concentrations of waste increase during the incubation period Parungao-Balolong 2011Thursday, July 14, 2011
  14. 14. THE LAG PHASE  No immediate increase in cell mass or cell number (Cell is synthesizing new components)  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 recover  Cells retool, replicate their DNA, begin to increase in mass and finally divide Parungao-Balolong 2011Thursday, July 14, 2011
  15. 15. THE LAG PHASE  LONG LAG PHASE ◦Inoculum is from an old culture ◦Inoculum is from a refrigerated source ◦Inoculation into a chemically- different medium  SHORT LAG PHASE (or even absent) ◦Young, vigorously growing exponential phase culture is transferred to fresh medium of same composition Parungao-Balolong 2011Thursday, July 14, 2011
  16. 16. EXPONENTIAL /LOG 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  Microorganism 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 moment Parungao-Balolong 2011Thursday, July 14, 2011
  17. 17. 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 products Parungao-Balolong 2011Thursday, July 14, 2011
  18. 18. 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  Death rate may decrease after the population has been drastically reduced due to resistant cells Parungao-Balolong 2011Thursday, July 14, 2011
  19. 19. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Measurement of Microbial Growth Culture Media Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  20. 20.  Temperature  Minimum growth temperature REQUIREMENTS  Optimum growth temperature FOR GROWTH:  Maximum growth temperature PHYSICAL Parungao-Balolong 2011Thursday, July 14, 2011
  21. 21. INFLUENCE OF LIPID CONTENT ◦PSYCHROPHILY HIGH CONTENT OF UNSATURATED FATTY ACIDS HELP MAINTAIN A SEMI-FLUID MEMBRANE STATE AT LOW TEMPERATURE ◦THERMOPHILY PROTEINS OR ENZYMES = INCREASED NUMBER OF SALT BRIDGES (RESIST UNFOLDING IN THE AQUEOUS MILIEU) MEMBRANES = RICH IN SATURATED FATTY ACIDS (STABLE AT HIGH TEMPERATURES) Parungao-Balolong 2011Thursday, July 14, 2011
  22. 22. TEMPERATURE RANGE  STENOTHERMAL MICROBES ◦Narrow range ◦Neisseria gonorrhea  EURYTHERMAL MICROBES ◦Wide range ◦Enterococcus faecalis Parungao-Balolong 2011Thursday, July 14, 2011
  23. 23. pH  Most bacteria grow between pH 6.5 and 7.5  Molds and yeasts grow between pH 5 and 6  Acidophiles grow in acidic environments Parungao-Balolong 2011Thursday, July 14, 2011
  24. 24. REQUIREMENTS FOR GROWTH: PHYSICAL  Osmotic pressure  Hypertonic environments, increase salt or sugar, cause plasmolysis  Extreme or obligate halophiles require high osmotic pressure  Facultative halophiles tolerate high osmotic pressure Parungao-Balolong 2011Thursday, July 14, 2011
  25. 25. REQUIREMENTS FOR GROWTH: PHYSICAL WATER SOURCE BACTERIA FUNGI ALGAE ACTIVITY 1.00 blood Most Gram none none (pure water) negative and non-halophiles 0.90 ham Most cocci and Fusarium, Mucor, Bacillus Rhizopus 0.60 Chocolate none Saccharomyces rouxii none 0.55 (DNA disordered) Parungao-Balolong 2011Thursday, July 14, 2011
  26. 26. REQUIREMENTS FOR GROWTH: PHYSICAL  1atm  BAROTOLERANT ◦Increased pressure does adversely affect them but not as much as it does non-tolerant bacteria  BAROPHILIC ◦Grow more rapidly at high pressures  TRIVIA: one barophile has been recovered from the Mariana trench near the Philippines (10, 500m depth) ◦Can only grow at pressure greater than 400-500 atm (at 2°C) Parungao-Balolong 2011Thursday, July 14, 2011
  27. 27. REQUIREMENTS FOR GROWTH: CHEMICAL  Carbon  Structural organic molecules, energy source  Chemoheterotrophs use organic carbon sources  Autotrophs use CO2 Parungao-Balolong 2011Thursday, July 14, 2011
  28. 28. REQUIREMENTS FOR GROWTH: CHEMICAL  Nitrogen  Trace elements  In amino acids and proteins  Inorganic elements required  Most bacteria decompose proteins in small amounts  Some bacteria use NH4+ or NO3–  Usually as enzyme cofactors  A few bacteria use N2 in nitrogen fixation  Sulfur  In amino acids, thiamine and biotin  Most bacteria decompose proteins  Some bacteria use SO42– or H2S  Phosphorus  In DNA, RNA, ATP, and membranes  PO43– is a source of phosphorus Parungao-Balolong 2011Thursday, July 14, 2011
  29. 29. REQUIREMENTS FOR GROWTH: CHEMICAL  Oxygen (O2) Parungao-Balolong 2011Thursday, July 14, 2011
  30. 30. REQUIREMENTS FOR GROWTH: CHEMICAL  Singlet oxygen: O2 boosted to a higher-energy state  Superoxide free radicals: O2–  Peroxide anion: O22–  Hydroxyl radical (OH•) Parungao-Balolong 2011Thursday, July 14, 2011
  31. 31. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Culture Media Measurement of Microbial Growth Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  32. 32.  Culture medium: CULTURE Nutrients prepared MEDIA for microbial growth  Sterile: No living microbes  Inoculum: Introduction of microbes into medium  Culture: Microbes growing in/on culture medium Parungao-Balolong 2011Thursday, July 14, 2011
  33. 33. CULTURE MEDIA  TYPES: Chemically-Defined and Complex  Chemically defined media: Exact chemical composition is known  Complex media: Extracts and digests of yeasts, meat, or plants  Nutrient broth  Nutrient agar Parungao-Balolong 2011Thursday, July 14, 2011
  34. 34. RECALL: HISTORY OF  BEFORE AGAR  GELATIN ◦Liquid medium ◦Frederick Loeffler ◦Meat extract  POTATO SLICES medium + gelatin ◦Robert Koch (1881) ◦But gelatin liquid at ◦Used boiled potato, room temperature sliced  AGAR ◦Not all bacteria grew well ◦Fannie Eilshemius Hesse (1882) ◦Agar used for jams and jelly Parungao-Balolong 2011Thursday, July 14, 2011
  35. 35. AGAR  Fannie, wife of Walther Hesse, was working in Kochs laboratory as her husbands technician and had previously used agar to  Complex polysaccharide  Used as solidifying agent for culture media in Petri plates, slants, and deeps  Generally not metabolized by microbes  Liquefies at 100°C Parungao-Balolong 2011Thursday, July 14, 2011
  36. 36. ANAEROBIC CULTURE METHODS  Reducing media  Contain chemicals (thioglycollate or oxyrase) that combine O2  Heated to drive off O2 Parungao-Balolong 2011Thursday, July 14, 2011
  37. 37. ANAEROBIC CULTURE METHODS Parungao-Balolong 2011Thursday, July 14, 2011
  38. 38. SELECTIVE MEDIA & DIFFERENTIAL MEDIA  SELECTIVE: Suppress unwanted microbes and encourage desired microbes.  DIFFERENTIAL : Make it easy to distinguish colonies of different Parungao-Balolong 2011Thursday, July 14, 2011
  39. 39. SELECTIVE MEDIA & DIFFERENTIAL MEDIA Parungao-Balolong 2011Thursday, July 14, 2011
  40. 40. ENRICHMENT MEDIA  Encourages growth of desired microbe  used when the population of your target microbe is low  used when your target microbe is damaged MRS = lactic acid bacteria Lactose Broth = enterics Parungao-Balolong 2011Thursday, July 14, 2011
  41. 41. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Culture Media Measurement of Microbial Growth Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  42. 42. MATHEMATICS OF GROWTH  GENERATION TIME ◦The time required for a microbial population to double in number  MEAN GROWTH RATE CONSTANT(k) ◦The rate of microbial population growth expressed in terms of the number of generations per unit time  MEAN GENERATION TIME (g) Parungao-Balolong 2011Thursday, July 14, 2011
  43. 43. DO THE MATH...  If 100 cells growing for 5 hours produced 1,720,320 cells: Parungao-Balolong 2011Thursday, July 14, 2011
  44. 44. MATHEMATICS OF GROWTH  N0 = initial population number  Nt = the population at time t  n = the number of generations in time t  Nt = N0 x 2n  To solve for n: ◦log Nt = log N0 + n ⋅ log 2 ◦n = log Nt – log N0 = log Nt – log N0 log 2 0.301 Parungao-Balolong 2011Thursday, July 14, 2011
  45. 45. SAMPLE COMPUTATION Parungao-Balolong 2011Thursday, July 14, 2011
  46. 46. SAMPLE COMPUTATION  Given an initial density of 4 x 104 Parungao-Balolong 2011Thursday, July 14, 2011
  47. 47. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106 Parungao-Balolong 2011Thursday, July 14, 2011
  48. 48. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time Parungao-Balolong 2011Thursday, July 14, 2011
  49. 49. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: Parungao-Balolong 2011Thursday, July 14, 2011
  50. 50. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: ◦t = 2 Parungao-Balolong 2011Thursday, July 14, 2011
  51. 51. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: ◦t = 2 ◦n = log (1 x 106) – log (4 x 104) Parungao-Balolong 2011Thursday, July 14, 2011
  52. 52. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: ◦t = 2 ◦n = log (1 x 106) – log (4 x 104) 0.301 Parungao-Balolong 2011Thursday, July 14, 2011
  53. 53. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: ◦t = 2 ◦n = log (1 x 106) – log (4 x 104) 0.301 ◦n = 4.65 Parungao-Balolong 2011Thursday, July 14, 2011
  54. 54. SAMPLE COMPUTATION  Given an initial density of 4 x 104  After 2 hours the cell density became 1 x 106  Compute for the generation time  Solution: ◦t = 2 ◦n = log (1 x 106) – log (4 x 104) 0.301 ◦n = 4.65 ◦Generation time = 2/4.65 or 0.43 hoursParungao-Balolong 2011 (t/n)Thursday, July 14, 2011
  55. 55. 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 cerevisiae Parungao-Balolong 2011Thursday, July 14, 2011
  56. 56. DIRECT MEASUREMENTS  Plate counts: Perform serial dilutions of a sample Direct methods  Plate counts  Filtration  Direct microscopic count  Dry weight Parungao-Balolong 2011Thursday, July 14, 2011
  57. 57. DIRECT MEASUREMENTS: Plate Count  Inoculate Petri plates from serial dilutions Parungao-Balolong 2011Thursday, July 14, 2011
  58. 58. DIRECT MEASUREMENTS: Plate Count  After incubation, count colonies on plates that have 25-250 or 30-300 colonies  report as (CFUs) Parungao-Balolong 2011Thursday, July 14, 2011
  59. 59. DIRECT MEASUREMENTS: Filtration Parungao-Balolong 2011Thursday, July 14, 2011
  60. 60. DIRECT MEASUREMENTS: Direct Microscopic Count Parungao-Balolong 2011Thursday, July 14, 2011
  61. 61. INDIRECT MEASUREMENTS: Turbidity Indirect methods  Turbidity  MPN  Metabolic activity  Dry weight Parungao-Balolong 2011Thursday, July 14, 2011
  62. 62. INDIRECT MEASUREMENTS: MPN  Multiple Tube Fermentation Test as measured in MPN or Most probable Number  Count positive tubes and compare to statistical MPN table. Parungao-Balolong 2011Thursday, July 14, 2011
  63. 63. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Measurement of Microbial Growth Culture Media Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  64. 64. PURE CULTURE  A pure culture contains only one species or strain.  A colony is a population of cells arising from a single cell or spore or from a group of attached cells.  A colony is often called a colony-forming unit (CFU). PURE Mixed Parungao-Balolong 2011Thursday, July 14, 2011
  65. 65. OBTAINING PURE CULTURE: Streak Plating Parungao-Balolong 2011Thursday, July 14, 2011
  66. 66. OBTAINING PURE CULTURE: Spread Plating Parungao-Balolong 2011Thursday, July 14, 2011
  67. 67. OBTAINING PURE CULTURE: Pour Plating Parungao-Balolong 2011Thursday, July 14, 2011
  68. 68. OBTAINING PURE CULTURE: Pour Plating Parungao-Balolong 2011Thursday, July 14, 2011
  69. 69. COLONY CHARACTERISTICS Parungao-Balolong 2011Thursday, July 14, 2011
  70. 70. OBTAINING PURE CULTURE: The Essentials  Julius Richard Petri (1887)  Easy to use, stackable (saving space), requirement for plating methods Parungao-Balolong 2011Thursday, July 14, 2011
  71. 71. POURING MEDIA ON YOUR DISHES Parungao-Balolong 2011Thursday, July 14, 2011
  72. 72. LECTURE OUTLINE Reproduction & Growth Requirements for Growth Physical Chemical Measurement of Microbial Growth Culture Media Obtaining Pure Cultures Preservation Methods Parungao-Balolong 2011Thursday, July 14, 2011
  73. 73. PRESERVATION METHODS: Long Term  Deep-freezing: –50°to –95°C  Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum Parungao-Balolong 2011Thursday, July 14, 2011
  74. 74. REVIVING LYOPHILIZED CULTURES http://www.jcm.riken.jp Parungao-Balolong 2011Thursday, July 14, 2011

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