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

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 Lag phase

Cells are growing at the maximum rate possible under given conditions Exponential phase Rate of growth is constant Population most uniform

Stationary phase Bacteria in stationary phase are usually at a concentration of 10 9 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

Decline in viable cells due to toxic wastes and nutrient depletion Death phase 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 log 10 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 mm 2 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/ mm 2 Chamber is 0.02 mm deep 700/0.02 = 700 x 50 = 3.5 x 10 4 cells/mm 3 = 3.5 x 10 7 cells/cm 3

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

Sterile media fed into vessel at same rate that media containing bacteria are removed Chemostat 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