1. BIOL 3340
Chapter 7

2. Chapter 7
Microbial Growth

3. Growth Increase in cellular constituents that may result in:
increase in cell number
e.g., when microorganisms reproduce by budding or binary fission
increase in cell size
e.g., coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions e.g aseptic fungi
microbiologists usually study population growth rather than growth of individual cells
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4. The Procaryotic Cell Cycle
Cell cycle is sequence of events from formation of new cell through the next cell division
most bacteria divide by binary fission
Two pathways function during cycle
DNA replication and partition
cytokinesis
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5. Figure 7.1: Binary Fission
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6. Chromosome Replication and Partitioning
Most procaryotic chromosomes are circular
Origin of replication – site at which replication begins
Terminus – site at which replication is terminated & is located directly opposite the origin
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7. ..Growth
Replisome:
Replisome is a group of proteins needed for DNA synthesis which assembled at the origin.
DNA replication then proceeds in both directions.
As daughter chromosomes are synthesised, the 2 newly formed origins moves towards opposite ends of the cell
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8. Figure 7.2:Cell Cycle of E.coli
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9. Figure 7.3-Cytoskeletal protein such as Actin homologue MreB , tubulin protein FTsZ & MinCD
Min protein.
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10. Cytoskeletal Proteins − Role in Cytokinesis
Cytokinesis is process not well understood
Protein MreB
similar to eucaryotic actin
plays a role in determination of cell shape and movement of chromosomes to opposite cell poles
Protein FtsZ
similar to eucaryotic tubulin
plays a role in Z ring formation which is essential for septation
MinCD-Min protein oscillates from pole to pole preventing an offcentre Z ring.
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11. …Growth
Cytokinesis is completed in several steps: 1. Selection of site where septum is formed 2. Assembly of the specialized structure, the Z ring which divides the cell into 2 by constriction 3. Linkage of Z ring to plasma membrane and may be other components of cell wall

12. ..Growth
4. Assembly of cell wall synthesizing machinery (e;g of peptidoglycan and other cell wall materials
5. Constriction of the cell wall accompanied by invagination of the cell membrane and septum formation.
Assembly of Z ring is crucial for other steps to follow.
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13. DNA Replication in Rapidly Growing Cells
A cell cycle is completed in 60 minutes e.g is a slow growing E coli.
40 minutes for DNA replication
20 minutes for septum formation and cytokinesis
Rapidly growing E. coli, a cell cycle may be completed in 20 mins.
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14. The Growth Curve
A growth curve is observed when microorganisms are cultivated in batch culture
A culture incubated in a closed vessel with a single batch of medium
usually plotted as logarithm of cell number versus time
usually has four distinct phases
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15. ..Growth Curve
Typically, a batch culture passes through four distinct stages:
Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage
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16. Figure 7.5: Microbial Growth Curve in a closed system
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17. Lag Phase Cell synthesizing new components Varies in length
e.g., to replenish spent materials
e.g., to adapt to new medium or other conditions
E.g cells may be old, synthesizes ATP, essential enzymes, ribosomes etc..or injured need time to recover
Varies in length
in some cases can be very short or even absent
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18. Exponential Phase Also called log phase
Rate of growth is constant and also called as balanced growth
Population is most uniform in terms of chemical and physical properties during this phase
Exponential growth- all cellular components are synthsized at constant rate
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19. Unbalanced Growth
Rates of synthesis of cell components vary relative to each other
occurs under a variety of conditions
change in nutrient levels
shift-up (poor medium to rich medium)
shift-down (rich medium to poor medium)
change in environmental conditions
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20. Balanced Growth During log phase, cells exhibit balanced growth
cellular constituents manufactured at constant rates relative to each other
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21. Effect of Nutrient Concentration on Growth
Figure 7.6 a): Nutrient concentration on total yield of microbial cells b) Nutrient on Growth rate
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22. ….Growth Curve Few similarities to passive diffusion:
Movement of substances across a membrane with the assistance of a transport protein
movement of molecules is not energy dependent
direction of movement is from high concentration to low concentration
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23. Stationary Phase
Total number of viable cells remains constant during lag phase
may occur because metabolically active cells stop reproducing
may occur because reproductive rate is balanced by death rate
Nutrient depletion e.g oxygen
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24. Possible Reasons for Entry into Stationary Phase
Nutrient limitation
Limited oxygen availability
Toxic waste accumulation
Critical population density reached
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25. Active Transport Energy-dependent process ( ATP)
Movement of substances across a membrane with the assistance of a transport protein (permeases).Active transport pumps are usually carrier proteins.
Energy-dependent process ( ATP)
Moves molecules against the concentration gradient i.e Movement of solutes from low  high concentration
Concentrates molecules inside cell.
E.g ATP/ABC Transporters
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26. Starvation Responses morphological changes
e.g., endospore formation
decrease in size, protoplast shrinkage, and nucleoid condensation
production of starvation proteins which makes cells much more resistant to damage. E;g increases in peptidodoglycan or DNA binding protein to protect DNA.
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27. …Starvation Responses
Protein Chaperone- to prevent protein denaturation
Hence starved cells difficult to kill, long-term survival ( for years) & increased virulence e.g Salmonella enterica
VBNC Viable but non-Culturable

28. Loss of Viability Figure 7.7: Loss of Viability
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29. The Mathematics of Growth
Generation (doubling) time
time required for the population to double in size
varies depending on species of microorganism and environmental conditions
range is from 10 minutes for some bacteria to several days for some eucaryotic microorganisms
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30. Figure 7.9: Exponential Microbial growth: Four generations of growth (red) plotted directly & in the logarithmic form (BlUe).
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31. The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in minutes
Growth rate (min-1) =
Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

32. Table 7.1 : 2 cells after 20 mins, 4 cells after 40 mins,, No =initial population number, n is the number of generations & Nt = Incubation time
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33. Measurement of Microbial Growth
can measure changes in number of cells in a population
can measure changes in mass of population
the most appropriate method depends on the experimental situation
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34. Measurement of Cell Numbers
Direct cell counts:
Counting chambers e.g Petroff-Hausser (Fig 7.11) for prokaryotes & Hemocytometers for pro and eucaryotes
Electronic counters for larger count such as yeast eg Flow cytometry
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35. Counting Chambers easy, inexpensive, and quick
useful for counting both eucaryotes and procaryotes
cannot distinguish living from dead cells
Figure 7.11-The Petroff-Hausser Counting Chamber
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36. Viable cell counts:
Plating methods- several plating method can be used to determine the number of viable microbes in a sample e.g spread plate and pour plate.
In both methods a diluted sample is dispersed over the media.
Membrane filters (fig.7.12)-

37. Plating Methods… simple and sensitive
widely used for viable counts of microorganisms in food, water, and soil
inaccurate results obtained if cells clump together
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38. ..membrane filter
In membrane filter method, bacterial cells are trapped from aquatic samples on a membrane filter.
Filter is then placed on agar media, incubated until colony is formed
Colony count is done

39. ..Viable count Method - Membrane filtration
especially useful for analyzing aquatic samples
Figure 7.12-The Membrane Filtration procedure
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40. Figure 7.13-Colonies on membranes
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41. Measurement of Cell Mass
Dry weight
time consuming and not very sensitive
Quantity of a particular cell constituent
e.g., protein, DNA, ATP, or chlorophyll
useful if amount of substance in each cell is constant
Turbidometric measures (light scattering)
quick, easy, and sensitive
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42. Figure 7.14-Microbial mass Measurement
more cells 
more light
scattered
less light
detected
Figure 7.14-Microbial mass Measurement
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43. The Continuous Culture of Microorganisms
Batch cultures in a closed system ( so far decribed) are those in which nutrients are replaced nor wastes are removed.
In a continous culture, growth in an open system
continual provision of nutrients
continual removal of wastes
maintains cells in log phase at a constant biomass concentration for extended periods
achieved using a continuous culture system e.g Chemostat
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44. …chemostat
A chemostat- the rate at which sterile medium is fed into the culture vessel, is the same as the rate at which media containing microorganisms is removed.
Chemostat contains essential nutrient e.g a vitamin in limiting quantities.

45. The Chemostat
rate of incoming medium = rate of removal of medium from vessel
an essential nutrient is in limiting quantities
Figure 7.15
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46. The Turbidostat
regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density by a photocell.
dilution rate varies
no limiting nutrient
turbidostat operates best at high dilution rates
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47. The Influence of Environmental Factors on Growth
most organisms grow in fairly moderate environmental conditions
Extremophiles
grow under harsh conditions that would kill most other organisms
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48. Solutes and Water Activity
water activity (aw):
amount of water available to organisms
reduced by interaction with solute molecules (osmotic effect)
higher [solute]  lower aw
reduced by adsorption to surfaces (matric effect)
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49. ..Water Activity (aw)
water activity of a solution is 1/100 the relative humidity of solution
also equal to ratio of vapor pressure (Psoln) of the solution to that of pure water (Pwater)
Aw = Psoln/ Pwater
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50. Osmotolerant Organisms
grow over wide ranges of water activity
many use compatible solutes to increase their internal osmotic concentration
compatible solutes are those which compatible with metabolism and growth
some have proteins and membranes that require high solute concentrations for stability and activity.
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51. Effects of NaCl on Microbial Growth
halophiles
grow optimally at >0.2 M, high level of NACL.
extreme halophiles
require >2 M e.g Halobacterium can grow in Dead Sea salt lake
Figure 7.17
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52. pH acidophiles neutrophiles alkalophiles
growth optimum between pH 0 and pH 5.5
neutrophiles
growth optimum between pH 5.5 and pH 8
alkalophiles
growth optimum between pH 8 and pH 11.5
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53. …pH
most acidophiles and alkalophiles maintain an internal pH near neutrality
some synthesize proteins that provide protection
e.g., acid-shock proteins
many microorganisms change pH of their habitat by producing acidic or basic waste products
most media contain buffers to prevent growth inhibition
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54. Temperature organisms exhibit distinct cardinal growth temperatures
minimal
maximal
optimal
Figure 7.19
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55. Figure 7.20
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56. Oxygen Concentration
Figure 7.21-Oxygen and Bacterial growth:SOD ( Enzyme superoxide dismutase) and catalase.
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57. Basis of Different Oxygen Sensitivities
oxygen easily reduced to toxic products
superoxide radical
hydrogen peroxide
hydroxyl radical
aerobes produce protective enzymes
superoxide dismutase (SOD) from toxic superoxide radical
Catalase-destroy hydrogen peroxide
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58. Pressure barotolerant barophilic organisms
adversely affected by increased pressure, but not as severely as nontolerant organisms
barophilic organisms
require or grow more rapidly in the presence of increased pressure (400 atm or greater)
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59. Radiation Damage ionizing radiation x rays and gamma rays
mutations  death
disrupts chemical structure of many molecules, including DNA
damage may be repaired by DNA repair mechanisms
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60. Radiation Damage… ultraviolet (UV) radiation mutations  death
causes formation of thymine dimers in DNA
DNA damage can be repaired by several repair mechanisms
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61. Microbial Growth in Natural Environments
microbial environments are complex, constantly changing, often contain low nutrient concentrations (oligotrophic environment) and may expose a microorganism to overlapping gradients of nutrients and environmental factors
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62. Responses to Low Nutrient Levels (oligotrophic environments)
organisms become more competitive in nutrient capture and use of available resources
morphological changes to increase surface area and ability to absorb nutrients
mechanisms to sequester certain nutrients
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63. Biofilms ubiquitous in nature
complex, slime enclosed colonies attached to surfaces than free floating.
when form on medical devices such as implants often lead to illness
can be formed on any conditioned surface
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64. …Biofilm Microorganisms
extracellular matrix and change in attached organisms’ physiology protects them from harmful agents such as UV light and antibiotics
sloughing off of organisms can result in contamination of water phase above the biofilm such as in a drinking water system
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65. Cell to Cell Communication
Acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms e.g marine luminescent Vibrio fisheri which glows.
AHL control the ability this bacterium to glow.
concentration present allows cells to access population density
process is called quorum sensing
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66. ..Quorum Sensing
AHL or other signal molecule diffuses across plasma membrane
at high concentrations it enters cell
once inside the cell it induces expression of target genes that regulate a variety of functions
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67. Fig Euprymna scolopes ( squid) buried day time and active at night. Light organ is covered with Vibrio fisheri
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68. Bibliography
Lecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Hill Co.