Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
MICROBIAL GROWTH 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 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 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 usually plotted as logarithm of cell number versus incubation time has four distinct phases ◦ lag, exponential, stationary, senescence, and death 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 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 during log phase, cells exhibit balanced growth ◦ cellular constituents manufactured at constant rates relative to each other 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 At sufficiently high nutrient concentration, transport system are saturated, growth rate does not rise further with increasing nutrient conc. 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 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 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 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 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 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 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 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 direct cell counts ◦ counting chambers ◦ on membrane filters haemocytometer easy, inexpensive, and quick useful for counting both eukaryotes and prokaryotes cannot distinguish living from dead cells 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 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 Count only those able to reproduce when cultured (plate count) Simple, sensitive Viable counting method - spread plate, pour plate - membrane filtration 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) 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 Measure cell mass can be used to follow growth dry weight turbidometric measures Dry weight Cell growing in liquid media – centrifuge, washed – dried in oven – weight Measure growth of filamentous fungi/ bacteria Time consuming, not very sensitive 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 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 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 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 Has photocell to measure turbidity flow rate of media automatically regulated to maintain a predetermined turbidity or cell density no limiting nutrient (nutrients in excess) Turbidostat maintain desired cell density 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 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 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 measure of the relative acidity of a solution negative logarithm of the hydrogen ion concentration 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 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 organisms exhibit distinct cardinal growth temperatures ◦ minimal ◦ maximal ◦ optimal 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 Microorganisms vary in their need for/tolerance of O2. aerobe ◦ grows in presence of atmospheric oxygen (O2) which is 20% O2 Obligate (strict) aerobe – requires O2 anaerobe ◦ grows in the absence of O2 obligate anaerobe ◦ usually killed in presence of O2 microaerophiles ◦ requires 2–10% O2 facultative anaerobes ◦ do not require O2 but grow better in its presence aerotolerant anaerobes ◦ grow with or without O2