1. Cell Growth and Binary Fission
Growth: increase in the number of cells
Binary fission: cell elongation following enlargement of a cell to twice its minimum size
Generation time: time required for a population of microbial cells to double
During cell division each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

2. Binary Fission in a Rod-Shaped Prokaryote
Figure 6.1

3. Growth Terminology and the Concept of Exponential Growth
Most bacteria have shorter generation times than eukaryotic microbes
Generation time is dependent on growth medium and incubation conditions
Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval
During exponential growth, the increase in cell number is initially slow but increases at a faster rate

4. The Rate of Growth of a Microbial Culture
Figure 6.8

5. Calculating Microbial Growth Parameters
Figure 6.9

6. The Mathematics of Exponential Growth
Increase in cell number in an exponentially growing bacterial culture is a geometric progression of the number 2
Relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth:
N = No2n
where N is the final cell number, No is the initial cell number, and n is the number of generations during the period of exponential growth

7. The Mathematics of Exponential Growth
Generation time (g) of the exponentially growing population is
g = t/n
where t is the duration of exponential growth and n is the number of generations during the period of exponential growth

8. The Mathematics of Exponential Growth
Specific growth rate (k) is calculated as
k = 0.301/g
Division rate (v) is calculated as
v = 1/g

9. The Microbial Growth Cycle
Batch culture: a closed-system microbial culture of fixed volume

10. Continuous Culture: The Chemostat
Continuous culture: an open-system microbial culture of fixed volume
Chemostat: most common type of continuous culture device
Both growth rate and population density of culture can be controlled independently and simultaneously
Dilution rate: rate at which fresh medium is pumped in and spent medium pumped out
Concentration of a limiting nutrient

11. Schematic for Continuous Culture Device (Chemostat)
Figure 6.11

12. Continuous Culture: The Chemostat
In a chemostat
The growth rate is controlled by dilution rate
The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient
In a batch culture growth conditions are constantly changing; it is impossible to independently control both growth parameters

13. The Effect of Nutrients on Growth
Figure 6.12

14. Continuous Culture: The Chemostat
Chemostat cultures are sensitive to the dilution rate and limiting nutrient concentration
Too high a dilution rate, the organism is washed out
Too low a dilution rate, the cells may die from starvation
Increasing limiting nutrient concentration results in greater biomass but same growth rate

15. Steady-State Relationships in the Chemostat
Figure 6.13

16. Measurements of Total Cell Numbers: Microscopic Counts
Microbial cells can be enumerated by microscopic observations; simple but results can be unreliable
Limitations of microscopic counts
Cannot distinguish between live and dead cells without special stains
Small cells difficult to see and can be overlooked
Precision is difficult to achieve
A phase-contrast microscope is required if a stain is not used

17. Measurements of Total Cell Numbers: Microscopic Counts
Limitations of microscopic counts (cont’d)
Cell suspensions of low density (< 106 cells/ml) hard to count
Motile cells need to immobilized
Debris in sample can be mistaken for cells
A second method for enumerating cells in liquid samples is with a flow cytometer

18. Direct Microscopic Counting Procedure
Figure 6.14

19. Viable Cell Counting
Viable cell counts (plate counts): measurement of living, reproducing population
Two main ways to perform plate counts
Spread-plate method
Pour-plate method
To obtain the appropriate colony number, the sample to be counted should always be diluted

20. Spread-Plate Method for the Viable Count
Figure 6.15

21. Pour-Plate Method for the Viable Count
Figure 6.15

22. Procedure for Viable Counting Using Serial Dilutions
Figure 6.16

23. Viable Cell Counting
Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (e.g., soil and water)
The Great Plate Anomaly: direct microscopic counts of natural samples typically reveal far more organisms than are recoverable on plates of any given culture medium

24. Viable Cell Counting Why is this?
Microscopic methods count dead cells whereas viable methods do not
Different organisms in even a very small sample may have vastly different requirements for resources and conditions in laboratory culture

25. Measurements of Microbial Mass: Turbidimetric Methods
Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth
Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.)

26. Turbidity Measurements of Microbial Growth
Figure 6.17a

27. Turbidity Measurements of Microbial Growth
To relate a direct cell count to a turbidity value, a standard curve must first be established
Figure 6.17b

28. Measurements of Microbial Mass: Turbidimetric Methods
Turbidity measurements
Quick and easy to perform
Typically do not require destruction or significant disturbance of sample
Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)

29. Effects of Temperature on Microbial Growth
Microorganisms can be classified into groups by their growth temperature optima
Psychrophile: low temperature
Mesophile: midrange temperature
Thermophile: high temperature
Hyperthermophile: very high temperature

30. Temperature and Growth Relations in Different Classes
Figure 6.19

31. Effects of Temperature on Microbial Growth
Mesophiles: organisms that have midrange temperature optima; found in
Warm-blooded animals
Terrestrial and aquatic environments
Temperate and tropical latitudes

32. Microbial Growth at Cold Temperatures
Extremophiles
Organisms that have evolved to grow optimally under very hot or very cold conditions
Psychrophiles
Organisms with cold temperature optima; the most extreme representatives inhabit permanently cold environments
Psychrotolerant
Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC; more widely distributed in nature than psychrophiles

33. Antarctic Microbial Habitats and Microorganisms
Figure 6.20a and b

34. Microbial Growth at Cold Temperatures
Molecular Adaptations to Psychrophily
Production of enzymes that function optimally in the cold; features that may provide more flexibility
More of -helices than -sheets
More polar and less hydrophobic amino acids
Fewer weak bonds
Decreased interactions between protein domains

35. Microbial Growth at Cold Temperatures
Molecular Adaptations to Psychrophily (cont’d)
Transport processes function optimally at low temperatures due to modifications of cytoplasmic membranes
High unsaturated fatty acid content

36. Microbial Growth at High Temperatures
Above ~65°C only prokaryotic life forms exist
Thermophiles: organisms with growth temperature optima between 45ºC and 80ºC
Hyperthermophiles: organisms with optima greater than 80°C
Inhabit hot environments including boiling hot springs and seafloor hydrothermal vents that can have temperatures in excess of 100ºC

37. Upper Temperature Limits for Growth of Living Organisms

38. Microbial Growth at High Temperatures
Hyperthermophiles in Hot Springs
Chemoorganotrophic and chemolithotrophic species present
High prokaryotic diversity (both Archaea and Bacteria represented)
Thermal gradients form along edges of hot environments
Distribution of microbial species along the gradient is dictated by organism’s biology

39. Growth of Hyperthermophiles in Boiling Water
Figure 6.22

40. Growth of Thermophilic Cyanobacteria in a Hot Spring
Figure 6.23

41. Microbial Growth at High Temperatures
Studies of thermal habitats have revealed
Prokaryotes are able to grow at higher temperatures than eukaryotes
Organisms with the highest temperature optima are Archaea
Nonphototrophic organisms can grow at higher temperatures than phototrophic organisms

42. Microbial Growth at High Temperature
Molecular Adaptations to Thermophily
Enzyme and proteins function optimally at high temperatures; features that provide thermal stability
Critical amino acid substitutions in a few locations provide more heat-tolerant folds
An increased number of ionic bonds between basic and acidic amino acids resist unfolding in the aqueous cytoplasm
Production of solutes (e.g., di-inositol phophate, diglyercol phosphate) help stabilize proteins

43. Microbial Growth at High Temperature
Hyperthermophiles and produce enzymes widely used in industrial microbiology
E.g., Taq polymerase; used to automate the repetitive steps in the polymerase chain reaction (PCR) technique

44. Microbial Growth at Low or High pH
The pH of an environment greatly affects microbial growth
Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)

45. The pH Scale
Figure 6.24

46. Microbial Growth at Low or High pH
Acidophiles: organisms that grow best at low pH (< 6)
Some obligate acidophiles; membranes destroyed at neutral pH
Stability of cytoplasmic membrane critical
Alkaliphiles: organisms that grow best at high pH (> 9)
Some have sodium motive force rather than proton motive force

47. Microbial Growth at Low or High pH
The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic
Internal pH has been found to be as low as 4.6 and high as 9.5 in extreme acido- and alkaliphiles, respectively

48. Oxygen and Microbial Growth
Aerobes: require oxygen to live
Anaerobes: do not require oxygen and may even be killed by exposure
Facultative organisms: can live with or without oxygen
Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cannot use it
Microaerophiles: can use oxygen only when it is present at levels reduced from that in air

49. Growth Versus Oxygen Concentration
Figure 6.27

50. Oxygen Relationships of Microorganisms

51. Incubation under Anoxic Conditions