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Microbial growth Typically refers to an increase in population rather than in size Growth curves Carried out using batch cultures or a closed system (no fresh media added) Characterized by several phases Lag phase Occurs when cells are placed into fresh media Likely due to the cells’ need to synthesize new components before reproducing Lag phase Can vary depending on: 1. Type of media 2. Condition of the cells Exponential phase Cells are growing at the maximum rate possible under given conditions Rate of growth is constant Population most uniform Stationary phase Bacteria in stationary phase are usually at a concentration of 109 cells per ml Balance between cell division and cell death or cells cease to divide Stationary phase Due to: Nutrient depletion Toxic waste accumulation Critical cell density reached Stationary phase Bacteria subjected to starvation may become resistant to killing Some pathogens may become more virulent when starved Death phase Decline in viable cells due to toxic wastes and nutrient depletion Death may be at a constant rate (logarithmic) Death rate may decrease after majority of population has died (resistant cells) Mathematics of growth Cells dividing at a constant rate during exponential growth Generation time/doubling time = time it takes for population to double Mathematics of growth More convenient to graph as log10 of cell number vs. time Generation time Determining generation time Measurement of microbial growth Measurement of cell number Measurement of cell mass Measurement of culture turbidity Measurement of cell number Counting chambers Coulter counters Plating techniques Membrane filter techniques Petroff-Hauser chamber Used for counting prokaryotic cells Use of stains or fluorescent or phase-contrast microscopes make counting easier Using a Petroff-Hauser chamber Chamber is of known depth and has grid etched into bottom 25 squares cover an area of 1 mm2 Determining average number per square and multiplying by 25 gives total number of cells in chamber Using a Petroff-Hauser chamber 280 cells in 10 squares 280/10 = 28/square 28 x 25 = 700 cells/ mm2 Chamber is 0.02 mm deep 700/0.02 = 700 x 50 = 3.5 x 104 cells/mm3 = 3.5 x 107 cells/cm3 Coulter counter Cells forced through small opening with electrodes on either side Passage of cell will cause resistance to increase and cell is counted More useful for counting eukaryotes Counting chambers and Coulter counters Neither can distinguish between living and dead cells Plating techniques Diluted sample spread over the surface of agar plate Number of cells can be calculated by multiplying colony number by dilution factor Membrane filter techniques Useful for measuring number of cells in aquatic samples Sample passed through filter with small pore size Filters placed on agar plates to allow growth of colonies Membrane filter techniques Measurement of dry weight Cells collected by centrifugation, washed and dried in an oven and weighed Most useful for fungi Measurement of turbidity Degree of light scattering induced by a culture is indirectly related to the cell number Spectrophotometers measure amount of light scattering Can measure transmittance or absorption of light Continuous culture of microorganisms Two most common systems Chemostat Turbidostat Chemostat Sterile media fed into vessel at same rate that media containing bacteria are removed Final cell density is dependant on the conc. of a limiting nutrient Turbidostat Makes use of a photocell to measure turbidity of culture Flow rate of media is regulated to maintain a constant cell density Influence of environmental factors on growth Influence of environmental factors on growth Influence of environmental factors on growth Influence of environmental factors on growth Acidophiles Neutrophiles Alkalophiles Influence of environmental factors on growth Influence of environmental factors on growth Quorum sensing Bacteria can communicate via quorum sensing or autoinduction Cell senses concentration of signal When threshold is reached, cell begins expressing sets of certain genes Quorum sensing Most common signal molecules in gram-negative bacteria are acyl homoserine lactones (HSLs) Gram-positives often use an oligopeptide signal molecule Important in pathogenicity and biofilm formation