1. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 Chapter 6 Microbial Growth

2. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 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 microbiologists usually study population growth rather than growth of individual cells

3. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 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

4. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 Binary fission

5. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 The Cell Cycle in E. coli E. coli requires ~40 minutes to replicate its DNA and 20 minutes after termination of replication to prepare for division

6. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 replisome – group of proteins needed for DNA synthesis; parent DNA spools through the replisome as replication occurs

7. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 Cell cycle of slow-growing E. coli

8. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 8 Cytoskeletal Proteins - Role in Cytokinesis 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

9. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 9 Cytoskeletal proteins involved in cytokinesis in rod-shaped bacteria MinCD, together with other Min proteins, oscillates from pole to pole : prevents the formation of an off-center Z ring

10. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 10 Formation of the cell division apparatus in E. coli

11. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11 DNA Replication in Rapidly Growing Cells cell cycle completed in 20 minutes –40 minutes for DNA replication –20 minutes for septum formation and cytokinesis look at timing - how can this happen? –second, third or fourth round of replication can begin before first round of replication is completed

12. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12 The Growth Curve Growth curve of microorganisms growing in a batch culture (closed system) –usually plotted as logarithm of cell number versus time –usually has four distinct phases –batch culture: culture incubated in a closed vessel with a single batch of medium

13. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 13 No increase in cell number Maximal rate of division and population growth Population growth ceases Decline in Population size

14. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 14 Lag phase cell synthesizing new components –e.g., to replenish spent materials –e.g., to adapt to new medium or other conditions varies in length –in some cases can be very short or even absent

15. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 15 Exponential phase also called log phase rate of growth is constant population is most uniform in terms of chemical and physical properties during this phase cells exhibit balanced growth –cellular constituents manufactured at constant rates relative to each other

16. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 16 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

17. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 Effect of nutrient concentration on growth Growth rate = number of generation/time (k = n/t)

18. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18 Stationary phase total number of viable cells remains constant –may occur because metabolically active cells stop reproducing –may occur because reproductive rate is balanced by death rate

19. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19 Possible reasons for entry into stationary phase nutrient limitation limited oxygen availability toxic waste accumulation critical population density reached

20. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 20 Starvation responses morphological changes –e.g., endospore formation decrease in size, protoplast shrinkage, and nucleoid condensation production of starvation proteins - long-term survival increased virulence in pathogenic bacteria

21. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 21 Death phase two alternative hypotheses on the declining of viable cells during death phase 1) cells are viable but nonculturable (VBNC) : cells alive, but dormant (← genetic response triggered in starving) 2) programmed cell death –fraction of the population genetically programmed to die (commit suicide)

22. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 22 Loss of viability

23. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 23 Prolonged decline in growth gradual decline instead of exponential decline in viability bacterial population continually evolves process marked by successive waves of genetically distinct varients natural selection occurs

24. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 The Mathematics of Growth generation (doubling) time (g) –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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26. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 26 Exponential growth

27. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 27 Generation time determination

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29. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 29 Measurement of Microbial Growth can measure changes in number of cells in a population can measure changes in mass of population

30. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 30 Measurement of Cell Numbers direct cell counts (total cell counts) –counting chambers –electronic counters –on membrane filters viable cell counts –plating methods –membrane filtration methods

31. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 31 Counting chambers easy, inexpensive, and quick useful for counting both eucaryotes and procaryotes cannot distinguish living from dead cells Petroff-Hausser counting chamber

32. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 32 Electronic counters Coulter counter and flow cytometer useful for large microorganisms and blood cells, but not procaryotes microbial suspension forced through small orifice movement of microbe through orifice impacts electric current that flows through orifice instances of disruption of current are counted

33. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 33 Direct counts on membrane filters cells filtered through special membrane that provides dark background for observing cells (black polycarbonate membrane filter) cells are stained with fluorescent dyes useful for counting bacteria with certain dyes, can distinguish living from dead cells

34. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 34 Viable Counting Methods spread and pour plate techniques –diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate –after incubation the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factor –results expressed as colony forming units (CFU)

35. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 35 Another viable count method - membrane filtration Especially useful for analyzing aquatic samples.

36. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 36 Colonies on membrane filters

37. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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

38. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 38 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

39. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 39 more cells  more light scattered  less light detected Absorbance (optical density)

40. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 40 The Continuous Culture of Microorganisms 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

41. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 41 The chemostat rate of incoming medium = rate of removal of medium from vessel an essential nutrient is in limiting quantities

42. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 42 Dilution rate and microbial growth dilution rate – rate at which medium flows through vessel relative to vessel size cell density remained unchanged over a wide range of dilution rates chemostat operates best at low dilution rate

43. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 43 The turbidostat regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density use a photocell to measure the absorbance or turbidity dilution rate varies no limiting nutrient (all nutrients are present in excess) turbidostat operates best at high dilution rates

44. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 44 Importance of continuous culture methods constant supply of cells in exponential phase growing at a known rate study of microbial growth at very low nutrient concentrations, close to those present in natural environment study of interactions of microbes under conditions resembling those in aquatic environments food and industrial microbiology

45. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 45 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

46. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 46

47. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 47 Solutes and water activity water activity (a w ) –amount of water available to organisms –reduced by interaction with solute molecules (osmotic effect) higher [solute]  lower a w – reduced by adsorption to surfaces (matric effect)

48. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 48 Water activity (a w ) water activity of a solution is 1/100 the relative humidity of solution also equal to ratio of solution’s vapor pressure (Psoln) to that of pure water (Pwater) a w = Psoln/ Pwater

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50. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 50 Osmotolerant organisms grow over wide ranges of water activity many use compatible solutes to increase their internal osmotic concentration –solutes that are compatible with metabolism and growth some have proteins and membranes that require high solute concentrations for stability and activity

51. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 51 Effects of NaCl on microbial growth halophiles –grow optimally at >0.2 M extreme halophiles –require >2 M

52. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 52 pH

53. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 53 pH acidophiles –growth optimum between pH 0 and pH 5.5 neutrophiles –growth optimum between pH 5.5 and pH 7 alkalophiles –growth optimum between pH 8.5 and pH 11.5

54. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 54 pH most acidophiles and alkalophiles maintain an internal pH near neutrality –the plasma membrane is impermeable to protons some extremophiles 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

55. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 55 Temperature organisms exhibit distinct cardinal temperatures –minimal –maximal –optimal

56. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 56

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58. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 58 Adaptations of thermophiles protein structure stabilized by a variety of means –e.g., more H bonds –e.g., more proline –e.g., chaperones histone-like proteins stabilize DNA membrane stabilized by variety of means –e.g., more saturated, more branched and higher molecular weight lipids –e.g., ether linkages (archaeal membranes)

59. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 59 Oxygen concentration need oxygen prefer oxygen ignore oxygen oxygen is toxic < 2 – 10% oxygen

60. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 60 Basis of different oxygen sensitivities oxygen easily reduced to toxic products –superoxide radical –hydrogen peroxide –hydroxyl radical aerobes produce protective enzymes –superoxide dismutase (SOD) –catalase

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63. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 63 GasPak anaerobic system

64. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 64 Pressure barotolerant –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

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66. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 66 Radiation Electromagnetic spectrum

67. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 67 Radiation damage ionizing radiation –cause atoms to lose electrons (ionize) –x-rays and gamma rays –mutations  death –disrupts chemical structure of many molecules, including DNA damage may be repaired by DNA repair mechanisms

68. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 68 Radiation damage… ultraviolet (UV) radiation –mutations  death –causes formation of thymine dimers in DNA –DNA damage can be repaired by several repair mechanisms

69. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 69 Radiation damage… visible light –at high intensities generates singlet oxygen ( 1 O 2 ) powerful oxidizing agent –carotenoid pigments protect many light-exposed microorganisms from photooxidation

70. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 70 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

71. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 71 Growth limitation by environmental factors Leibig’s law of the minimum –total biomass of organism will be determined by nutrient present at lowest concentration Shelford’s law of tolerance –above or below certain environmental limits (e.g., temperature), a microorganism will not grow, regardless of the nutrient supply

72. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 72 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

73. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 73 Morphology and nutrient absorption (Caulobacter) (a)Stalks are relatively short when phosphorus is plentiful. (b)The stalks are extremely long under phosphorus-limited conditions.

74. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 74 Counting viable but nonculturable vegetative procaryotes stressed microorganisms can temporarily lose ability to grow using normal cultivation methods microscopic and isotopic methods for counting viable but nonculturable cells have been developed

75. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 75 Counting and identifying microbes in natural environments –difficult to culture organisms from natural environments –previously stressed microbes are very sensitive to secondary stresses and may not grow on media normally used to cultivate them –Postgate microviability assay uses change in morphology in a thin agar film under a coverslip as indication of “life signs”

76. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 76 Biofilms ubiquitous in nature complex, slime enclosed colonies attached to surfaces when form on medical devices such as implants often lead to illness can be formed on any conditioned surface

77. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 77 (a)Biofilm on the surface of a stromatolite. The film consists primarily of the cyanobacterium Calothrix. (b)Biofilm (white color) on the surface of artificial joint

78. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 78 Biofilm formation microbes reversibly attach to conditioned surface and release polysaccharides, proteins, and DNA additional polymers are produced as biofilm matures interactions occur among the attached organisms

79. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 79 Biofilm formation

80. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 80 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

81. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 81 Cell to cell communication acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms AHL or other signal molecule diffuses across plasma membrane AHL diffuses out of the cell at low cell density but enters the cell when the cell population increases and AHL accumulates outside the cell once inside the cell it induces expression of target genes that regulate a variety of functions

82. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 82 Quorum sensing influx of AHL is cell-density-dependent concentration present allows cells to access population density - process is called quorum sensing

83. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 83 Representative cell-cell communication molecules