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