1. Growth & Culturing
of Bacteria
Microbiology 130
Chapter 6

2. Microbial Growth and Cell Division
Microbial Growth Defined:
Microbial growth is the increase in number of cells, not cell size
Mother cells
Daughter cells
Cell Division:
Binary fission
Tetrads
Sarcinae
Budding

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

4. Binary Fission

5. Phases of Growth 4 Phases: 1) Lag phase- 2) Log (logarithmic) phase
3) Stationary phase
4) Decline phase or death phase

6. Log Phase

7. Log Phase with Calculations
If 100 cells growing for 5 hours produced 1,720,320 cells:

8. Log phase

9. 4 Phases of Microbial Growth

10. Growth in Colonies 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).

11. Measuring Bacterial Growth Serial Dilutions
Direct Measurements of Microbial Growth
Plate counts: Perform serial dilutions of a sample

12. Standard Plate Count Inoculate Petri plates from serial dilutions
2 methods:
Pour Plate
Spread Plate

13. Plate Count
After incubation, count colonies on plates that have colonies (CFUs)

14. Direct Measurements of Microbial Growth
Filtration

15. Direct Measurements of Microbial Growth
Multiple tube MPN test.
Count positive tubes and compare to statistical MPN table.

16. Direct Measurements of Microbial Growth
Direct microscopic count

17. Most Probable Number Estimate of number
MPN 5 test tubes 10, 1, and 0.1 ml
Growth is displayed by production of gas bubbles or by becoming cloudy
Estimates are from a known chart ( table 6.1 pg 149)

18. Direct Measurements of Microbial Growth

19. Other Methods: Estimating Bacterial Numbers by Indirect Methods
Turbidity

20. Measuring Microbial Growth
Direct methods
Plate counts
Filtration
MPN
Direct microscopic count
Dry weight
Indirect methods
Turbidity
Metabolic activity

21. Factors Affecting Bacterial Growth. The Requirements for Growth:
Factors Affecting Bacterial Growth The Requirements for Growth: Physical Requirements
Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature

22. Temperature
Figure 6.1

23. Psychrotrophs/Psychrophiles
Grow between 0°C and 20-30°C
Cause food spoilage

24. Psychrotrophs/Psychrophiles
Figure 6.2

25. 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

26. Mesophiles & Thermophiles
grow best between 25 degrees C and 40 degrees C
Thermophiles-
Heat loving- grow best at degrees C
Obligate thermophiles
Facultative thermophiles-

27. The Requirements for Growth: Physical Factors - Chemical Requirements
Oxygen (O2)

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

29. The Requirements for Growth: Physical Factors / 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

30. The Requirements for Growth: Physical Requirements
Figure 6.4

31. The Physical Requirements for Growth
Moisture-
Hydrostatic Pressure-
Radiation-

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

33. The Requirements for Growth: Nutritional Factors - Chemical Requirements
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3–
A few bacteria use N2 in nitrogen fixation
Sulfur
In amino acids, thiamine and biotin
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus

34. The Requirements for Growth: Nutritional Factors - Chemical Requirements
Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors
Vitamins- organic substances and growth factors
Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines
Nutritional Complexity
Locations of Enzymes
Adaptations to Limited Nutrients

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

36. Sporulation / Endospores
Formation of endospores in Bacillus, Clostridium
and G+ genera
Can Survive long periods of drought
Axial nucleoid
Endospore septum grows
Spore coat and Exosporium
Germination- 3 stages:
1) Activation, 2) germination, 3) outgrowth

37. Culturing Bacteria Methods of Obtaining Pure Culture
1) The Streak Plate Method – sterile wire loop and streaked in different patterns on agar
2) Pour Plate Method- serial dilutions using melted agar

38. Streak Plate

39. 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
Synthetic media
Defined synthetic media
Complex media

40. 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

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

42. Culture Media
Tables 6.2, 6.4

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

44. Anaerobic Culture Methods
Anaerobic jar
Figure 6.5

45. Anaerobic Culture Methods
Anaerobic chamber
Figure 6.6

46. Capnophiles Require High CO2
Candle jar
CO2-packet
Figure 6.7

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

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

49. 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
Only phenol-metabolizing bacteria will be growing

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

51. Methods of Performing Multiple Diagnostic Tests
Enterotube Multitest API- simultaneous testing
To identify enteric bacteria- cause food poisoning, typhoid, shigellosis, gastroenteritis etc
Living, But Non-culturable organisms:
Some microbs can be observed with microscope, identify their DNA but they can not be cultured