Essential Factors for Microbial Growth in Bacteria Cultivation

1. 6 Microbial Growth

2. Microbial Growth • Microbial growth is the increase in number of cells, not cell size

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

4. Temperature Figure 6.1

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

6. Psychrotrophs Figure 6.2

7. Figure 6.3

8. 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

9. The Requirements for Growth: Physical Requirements • Osmotic pressure • Hypertonic environments, increase salt or sugar, cause plasmolysis • Extreme or obligatehalophiles require high osmotic pressure – up to 30% salt • Facultative halophiles tolerate high osmotic pressure – up to 2% salt

10. Plasmolysis in a Plant Cell

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. The Requirements for Growth: Chemical Requirements • Nitrogen • In amino acids and proteins • Some bacteria use NH4+ or NO3– • 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

14. The Requirements for Growth: Chemical Requirements • Oxygen (O2) Table 6.1

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

16. The Requirements for Growth: Chemical Requirements • Organic growth factors • Organic compounds obtained from the environment • Vitamins, amino acids, purines, and pyrimidines

17. 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

18. 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

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

20. Culture Media Tables 6.2, 6.4

21. Anaerobic Culture Methods • Anaerobic chamber Figure 6.6

22. Capnophiles Require High CO2 • Candle jar • CO2-packet Figure 6.7

23. Selective Media • Suppress unwanted microbes and encourage desired microbes. Figure 6.9b–c

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

25. 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

26. 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).

27. Streak Plate Figure 6.10a–b

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

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

30. Binary Fission Figure 6.11

31. Figure 6.12b

32. If 100 cells growing for 5 hours produced 1,720,320 cells: PLAY Animation: Bacterial Growth

33. Figure 6.13

34. Phases of Growth Figure 6.14

35. Direct Measurements of Microbial Growth • Plate counts: Perform serial dilutions of a sample Figure 6.15, step 1

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

37. Figure 6.16 - Overview

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

39. Direct Measurements of Microbial Growth • Filtration Figure 6.17

40. Direct Measurements of Microbial Growth • Direct microscopic count

41. Direct Measurements of Microbial Growth Figure 6.19, steps 1, 3