1. Chapter 6
Microbial Growth

2. Microbial Growth
Microbial growth = 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. The Requirements for Growth: Physical Requirements
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
Acidophiles grow in acidic environments

8. 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

9. The Requirements for Growth: Physical Requirements
Figure 6.4

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

11. The Requirements for Growth: Chemical Requirements
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
Some bacteria use SO42 or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43 is a source of phosphorus

12. The Requirements for Growth: Chemical Requirements
Trace Elements
Inorganic elements required in small amounts
Usually as enzyme cofactors

13. The Requirements for Growth: Chemical Requirements
Oxygen (O2)
obligate aerobes
Faultative anaerobes
Obligate anaerobes
Aerotolerant anaerobes
Microaerophiles

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

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

16. 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

17. 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

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

19. Culture Media
Table 6.2 & 6.4

20. Culture Media

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

22. Anaerobic Culture Methods
Anaerobic jar
Figure 6.5

23. Anaerobic Culture Methods
Anaerobic chamber
Figure 6.6

24. Capnophiles require high CO2
Candle jar
CO2-packet
Figure 6.7

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

26. Selective Media Used for growth of only selected microorganisms.
Selection by:
Adding antibiotics, prevents the growth of other cells
Lacking amino acids
May contain stains and color indicators (EMB)

27. Selective Media 1- Eosin-methylene blue agar (EMB)
Contains methylene blue, toxic to gram positive bacteria, allowing only the growth of gram negative bacteria
2- MacConkey agar (MCK)
Permit growth of gram negative bacteria and prevent gram positive due to presence of crystal violet and bile salts.

28. Selective Media 3- Mannitol salt agar (MSA)
Selective for Staphylococci
4- Thiosulfate citrate bile sucrose (TCBS)
Vibrio cholerae

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

30. Differential Media
Distinguishes one microorganism type from another growing on the same media on a difference in the colony appearance (Color and shape)
1- MacConkey agar (MCK)
Differential for lactose fermentation
2- Mannitol salt agar (MSA)
Differential for mannitol fermentation

31. Differential Media

32. Differential Media
MacConkey
agar

33. Enrichment Media Encourages growth of desired microbe
Use for cultivation of fastidious organisms such as Haemophilus influenza and Streptococcus spp
Enriched with blood, milk, serum, egg and vitamines.

34. Enrichment Media

35. Hemolysis with blood agar

36. Staphylococcus aureus on chocolate agar

37. 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)

38. Streak Plate
Figure 6.10a, b

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

40. lyophilizer

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

42. Reproduction in Prokaryotes

43. Binary Fission
Figure 6.11

44. Figure 6.12b

45. Figure 6.13

46. Figure 6.14

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

48. Plate Count
Inoculate Petri plates from serial dilutions
Figure 6.16

49. Plate Count
After incubation, count colonies on plates that have colonies (CFUs)
Figure 6.15

50. Direct Measurements of Microbial Growth
Filtration

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

52. Direct Measurements of Microbial Growth
Most probable
number method

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

54. Direct Measurements of Microbial Growth
Direct Microscopic Count

55. Direct Measurements of Microbial Growth
Petroff hausser cell counter
Figure 6.19

56. Estimating Bacterial Numbers by Indirect Methods

57. Estimating Bacterial Numbers by Indirect Methods
Turbidity
Figure 620

58. Estimating Bacterial Numbers by Indirect methods
Metabolic activity
As bacteria in media, they produce certain products such as carbon dioxide and acids.
Measure metabolic products.
Expensive.

59. Estimating Bacterial Numbers by Indirect methods
Dry weight
Bacteria and fungi in liquid media are centrifuged
Resulting cell pellet is weighted
Doesn't distinguish live and dead cells.