ERT 107

1. ERT 107 MICROBIAL GROWTH

2. Reproductive Strategies • The reproductive strategies of eukaryotic microbes • asexual (mitosis) and sexual (meiosis) • Bacteria and Archaea • asexual - binary fission, budding, filamentous • all must replicate and segregate the genome prior to division

5. Growth • increase in cellular constituents that may result in: • increase in cell number • increase in cell size • growth refers to population growth rather than growth of individual cells

6. The Growth Curve • Population growth – studied by analyzing the growth curve of a microbial culture • observed when microorganisms are cultivated in batch culture • culture incubated in a closed vessel with a single batch of medium • No in n out fresh medium, nutrient conc decline, waste increase

7. usually plotted as logarithm of cell number versus incubation time • has four distinct phases • lag, exponential, stationary, senescence, and death

9. Lag phase • Microorganism introduced into fresh medium • No increase in cell number • cell synthesizing new components • to replenish spent materials • to adapt to new medium or other conditions • time to recover; cell may be injured • varies in length • in some cases can be very short or even absent

10. Exponential phase • also called log phase • Microorganisms are growing & dividing at max rate • rate of growth and division is constant and maximal (completing cell cycle,doubling) • population is most uniform in terms of chemical and physical properties during this phase

11. during log phase, cells exhibit balanced growth • cellular constituents manufactured at constant rates relative to each other

12. Unbalanced growth • rates of synthesis of cell components vary relative to each other until balanced state is reached • 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

13. At sufficiently high nutrient concentration, transport system are saturated, growth rate does not rise further with increasing nutrient conc.

14. Stationary phase • closed system population growth eventually ceases, growth curve become horizontal • Happened at population level around 109 cell per ml • Final population size depend on nutrient availability, type of microorganisms • total viable cell number remain constant • active cells stop reproducing or reproductive rate is balanced by death rate

15. Possible Reasons for Stationary Phase • nutrient limitation (nutrient deplete, population growth will slow) • limited oxygen availability (O2 deplete, only surface have adequate O2) • toxic waste accumulation (byproduct toxic to microbe) • critical population density reached

16. Stationary Phase andStarvation Response • entry into stationary phase due to starvation and other stressful conditions activates survival strategy • morphological changes • e.g., endospore formation • decrease in size, protoplast shrinkage, and nucleoid condensation • RpoS protein assists RNA polymerase in transcribing genes for starvation proteins

17. Starvation Responses • production of starvation proteins • increase cross-linking in cell wall • Dps protein protects DNA • chaperone proteins prevent protein damage • cells are called persister cells • long-term survival • increased virulence

18. Senescence and Death Phase • No of viable cell decline at exponential rate • Nutrient deprivation, buildup of toxic waste cause irreparable harm to cell • No cellular growth if transfer to fresh medium • Because loss viability not accompany by loss in total cell no. assume that cell died but not lyse

19. two alternative hypotheses: a) cells are Viable But Not Culturable (VBNC) • Cells are unable to grow temporarily • cells alive, but dormant, capable of new growth when conditions are right b) programmed cell death • fraction of the population genetically programmed to die (commit suicide) • Some cell die, nutrient they leak enable growth of other cell • Altruistic – they sacrifice themselves for benefit of others

21. The Mathematics of Growth • Exponential phase – microbe dividing at constant interval • 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 eukaryotic microorganisms

23. Measurement of Growth Rate and Generation Time

24. Measurement of Microbial Growth • Many ways to measure growth rate and generation time • can measure changes in number of cells in a population • can measure changes in mass of population

25. Direct Measurement of Cell Numbers • direct cell counts • counting chambers • on membrane filters

26. Counting Chambers • haemocytometer • easy, inexpensive, and quick • useful for counting both eukaryotes and prokaryotes • cannot distinguish living from dead cells

27. On bottom of chamber is an etched grid to facilitate counting the cells • Number of microbe calculated by taking into account the chamber’s volume and dilution made

28. Direct Counts on Membrane Filters • Sample is filtered through black polycarbonate membrane that provides dark background for observing cells • cells are stained with fluorescent dyes • Observed microscopically and count • useful for counting bacteria (aquatic sample) • with certain dyes, can distinguish living from dead cells

29. Viable Counting Methods • Count only those able to reproduce when cultured (plate count) • Simple, sensitive • Viable counting method - spread plate, pour plate - membrane filtration

30. spread and pour plate techniques • diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate • Cell with grow as distinct colony • 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)

31. membrane filter technique • bacteria from aquatic samples are trapped on membranes of known pore size • membrane soaked in culture media • colonies grow on membrane • colony count determines number of bacteria in original sample

33. Indirect Measurement of Cell Mass • Measure cell mass can be used to follow growth • dry weight • turbidometric measures

34. Dry weight • Cell growing in liquid media – centrifuge, washed – dried in oven – weight • Measure growth of filamentous fungi/ bacteria • Time consuming, not very sensitive

35. turbidometric measures • Micobial cell scatter light that strike them • Amount of scattering proportional to the biomass of cell present • Extent of light scattering can be measure using spectrophotometer (absorbance) • Increase cell concentration, greater turbidity, more light scattered and absorbance reading will increase

37. The Continuous Culture of Microorganisms • Batch culture(closed system) - nutrient not renewed - waste not removed - exponential growth last only for few generation • growth in an open system (continuous culture system) - continual provision of nutrients • continual removal of wastes • maintains cells in log phase at a constant biomass concentration for extended periods

38. 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

39. The Chemostat • rate of incoming medium = rate of removal of medium containing microorganisms from vessel • an essential nutrient is in limiting quantities • Growth rate determined by rate at which fresh medium fed into chamber

40. The Turbidostat • Has photocell to measure turbidity • flow rate of media automatically regulated tomaintain a predetermined turbidity or cell density • no limiting nutrient (nutrients in excess) • Turbidostat maintain desired cell density

41. 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 Solute & water activity, pH, temperature, oxygen level

43. Solutes and Water Activity • changes in osmotic concentrations in the environment may affect microbial cells • hypotonic solution (lower osmotic concentration) • water enters the cell • cell swells may burst • hypertonic (higher osmotic concentration) • water leaves the cell • membrane shrinks from the cell wall (plasmolysis) may occur

44. Extremely Adapted Microbes • halophiles • grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M • extreme halophiles • require salt concentrations of 2M and 6.2M • extremely high concentrations of potassium • cell wall, proteins, and plasma membrane require high salt to maintain stability and activity

45. pH • measure of the relative acidity of a solution • negative logarithm of the hydrogen ion concentration

46. acidophiles • growth optimum between pH 0 and pH 5.5 • neutrophiles • growth optimum between pH 5.5 and pH 7 • alkaliphiles (alkalophiles) • growth optimum between pH 8.5 and pH 11.5

47. Temperature • Temp affect living organisms in 2 ways: - temp rise, chemical & enzymatic reaction rise - at very high temp, particular protein may damage • enzymes have optimal temperature at which they function optimally • high temperatures may inhibit enzyme functioning

48. organisms exhibit distinct cardinal growth temperatures • minimal • maximal • optimal

49. Temperature Ranges for Microbial Growth • psychrophiles – 0o C to 20o C • psychrotrophs – 0o C to 35o C • mesophiles – 20o C to 45o C • thermophiles – 55o C to 85o C • hyperthermophiles – 85o C to 113o C