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Chapter 6

Chapter 6. Microbial Growth. Microbial growth = increase in number of cells, not cell size. What are. Populations Colonies. The Requirements for Growth. Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements

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Chapter 6

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  1. Chapter 6 Microbial Growth

  2. Microbial growth = increase in number of cells, not cell size

  3. What are • Populations • Colonies

  4. The Requirements for Growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor

  5. The Requirements for Growth: Physical Requirements • Temperature • Minimum growth temperature • Optimum growth temperature • Maximum growth temperature

  6. Temperature Figure 6.1

  7. Psychrotrophs • Grow between 0°C and 20-30°C • Cause food spoilage

  8. Psychrotrophs Figure 6.2

  9. The Requirements for Growth: Physical Requirements • 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

  10. The Requirements for Growth: Physical Requirements • Osmotic Pressure • Hypertonic environments, increase salt or sugar, cause plasmolysis • Extreme or obligate halophiles require high osmotic pressure • Facultative halophiles tolerate high osmotic pressure

  11. The Requirements for Growth: Physical Requirements Figure 6.4

  12. The Requirements for Growth: Chemical Requirements • Carbon • Structural organic molecules, energy source • Chemoheterotrophs use organic carbon sources • Autotrophs use CO2

  13. Nitrogen • In amino acids, proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3 • A few bacteria use N2 in nitrogen fixation • Sulfur • In amino acids, thiamine, biotin • Most bacteria decompose proteins • Some bacteria use SO42 or H2S • Phosphorus • In DNA, RNA, ATP, and membranes • PO43is a source of phosphorus

  14. Trace Elements • Inorganic elements required in small amounts • Usually as enzyme cofactors

  15. Oxygen (O2)

  16. Toxic Forms of Oxygen • Singlet oxygen: O2 boosted to a higher-energy state • Superoxide free radicals: O2 • Peroxide anion: O22 • Hydroxyl radical (OH)

  17. Biofilms Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensing Figure 6.5

  18. The Requirements for Growth: Chemical Requirements • Organic Growth Factors • Organic compounds obtained from the environment • Vitamins, amino acids, purines, pyrimidines

  19. Culture Media • Culture Medium: Nutrients prepared for microbial growth • Sterile: No living microbes • Inoculum: Introduction of microbes into medium • Culture: Microbes growing in/on culture medium

  20. Agar • Complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies ~40°C

  21. Culture Media • Chemically Defined Media: Exact chemical composition is known • Complex Media: Extracts and digests of yeasts, meat, or plants • Nutrient broth • Nutrient agar

  22. Culture Media Table 6.2 & 6.4

  23. Anaerobic Culture Methods • Reducing media • Contain chemicals (thioglycollate or oxyrase) that combine O2 • Heated to drive off O2

  24. An Anaerobic Chamber Figure 6.7

  25. Anaerobic Culture Methods • Anaerobic jar Figure 6.5

  26. Capnophiles require high CO2 • Candle jar • CO2-packet Figure 6.7

  27. 1: No special precautions 2: Lab coat, gloves, eye protection 3: Biosafety cabinets to prevent airborne transmission 4: Sealed, negative pressure Exhaust air is filtered twice Biosafety Levels

  28. Biosafety Level 4 (BSL-4) Laboratory Figure 6.8

  29. Selective Media • Suppress unwanted microbes and encourage desired microbes. Figure 6.9b, c

  30. Differential Media • Make it easy to distinguish colonies of different microbes. Figure 6.9a

  31. Enrichment Media • Encourages growth of desired microbe • Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria • Inoculate phenol-containing culture medium with the soil and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Only phenol-metabolizing bacteria will be growing

  32. 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)

  33. Streak Plate Figure 6.10a, b

  34. Preserving Bacteria Cultures • Deep-freezing: -50°to -95°C • Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum

  35. Reproduction in Prokaryotes • Binary fission • Budding • Conidiospores (actinomycetes) • Fragmentation of filaments

  36. Binary Fission Figure 6.11

  37. Figure 6.12b

  38. If 100 cells growing for 5 hours produced 1,720,320 cells:

  39. Figure 6.13

  40. Phases of Growth ANIMATION Bacterial Growth Curve Figure 6.15

  41. Direct Measurements of Microbial Growth • Plate Counts: Perform serial dilutions of a sample Figure 6.15, top portion

  42. Plate Count • Inoculate Petri plates from serial dilutions Figure 6.16

  43. Plate Count • After incubation, count colonies on plates that have 25-250 colonies (CFUs) Figure 6.15

  44. Direct Measurements of Microbial Growth • Filtration Figure 6.17a, b

  45. Direct Measurements of Microbial Growth • Multiple tube MPN test • Count positive tubes and compare to statistical MPN table. Figure 6.18b

  46. Direct Measurements of Microbial Growth • Direct Microscopic Count

  47. Direct Measurements of Microbial Growth Figure 6.19

  48. Estimating Bacterial Numbers by Indirect Methods • Turbidity Figure 620

  49. Estimating Bacterial Numbers by Indirect methods • Metabolic activity • Dry weight

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