L15: Bacterial Growth And Measurement Of Growth Flashcards
Bacterial binary fission
Bacterial reproduction
-> pop increase in number (logarithmic)
Generation time (g)
The time for cell to produce 2 cells or time for bacterial pop to double in no.
As no. of bacteria increases in environment..
Nutrients used up
Metabolic wastes that may be toxic accumulate
Living space may become limited
Aerobes may suffer from oxygen depletion
-> bacterial growth stops
Significance of bacterial growth
Has human health implications: bacterial meningitis, cholera, food poising from staphylococci or salmonella, plague, tetanus etc
Has animal health implications: anthrax in ruminants, mastitis in dairy herds etc
Has environmental impacts: eutrophication (enrichment of aquatic environment with nutrients e.g. from sewage pollution), blooms of toxic cyanobacteria, acid sulphate soils, composting and sewage treatment
Provides for industrial applications: producing products (e.g. cheese, antibiotics, insulin), industrial processes (e.g. leaching metals from low grade ores)
Stationary cells
Non-dividing cells inoculated into fresh medium and begin to grow and multiply
Phases in bacterial growth in culture
Lag phase -> exponential or log phase -> stationary phase -> death phase
Y axis: log number of viable cells
X axis: time
- Lag phase
Inoculate stationary cells
Cells have to adapt to new physiological conditions
Synthesis of cellular constituents (ribosomes & enzymes) begins
Energy (ATP) become available for growth
Cell volume increases but no cell division-> growth rate (k) and generation time (g)= 0
Rates of physiological process, initially unbalanced -> different genes are upregulated at different times
DNA replication and cell division initiated (cell division and hence pop growth begins)
- Exponential (or Log) Phase
Bacteria are growing and dividing at max rate possible for particular strain , medium, and environment
Growth: balanced - relative synthesis rates are constant (DNA, ribosomes, enzymes, cell walls etc)
Growth rate (or growth rate constant). k=no. of generations per unit time, often per hour
k and g are constant
Graph of log [no.] vs. time produces straight line (slope = k)
Rate of growth depends in conditions (temp, nutrients). Different (stable) conditions -> exponential growth occurs but at different rate
Exponential growth continues until some factor becomes limiting. Limiting factors-> drive culture into stationary phase
- Stationary phase of growth
Total viable cell numbers remain constants (k=0): cells are not dividing (nutrients or O2 have been depleted, but remain metabolically active) or metabolic waste/toxin may have built up and cell death or inactivation balances cell division
Properties of cultures in stationary phase
Cells tend to be small
Cells have different composition of cellular constituents compared to growing cells
Growth again unbalanced
Available energy used for maintenance (cell survival mechanisms e.g. endospore formation may be initiated)
- Cell death phase
Cells begin to die (nutrient deprivation or toxic waste build up -> irreplaceable damage to cells)
K becomes -ve
Pop death rate usually logarithmic (constant proportion of cell dies per unit time)
Hypotheses of cells in death phase
‘Classical view’: even when cells transferred to fresh medium, did not resume growth. Assumed cells died but did not lyse
Other hypotheses:
- Do bacteria in death phase actually become ‘viable but not nonculturable’ cells? These are dormant cells, could possibly resume growth
- Does a fraction of pop of cells die due to activation of programmed cell death genes? Dead cells then release nutrients which support growth of other cells. Dying cells sacrifice themselves for rest
- Long term stationary phase
Some exhibit this phase
Can last months
Pop. continually evolves, actively growing cells use nutrients released by dying cells
Successive waves of genetically distinct variants
What about bacterial pop growth in natural world?
Bacteria in soils, waters, surfaces, intestinal tracts: subject to stimulants and inhibitors of growth
Natural systems: complex. Variable in physical, chemical and bio factors affecting pop.
Many microbes form biofilms: aggregations of microbes in complex communities, growing on surfaces and held together by extracellular polymers
Growth cycles fluctuate to reflect environmental variability and complexity
How is bacterial growth measured?
Measure cell biomass
Measure cell no.
Measure cell biomass
Dry weight determination: cells in broth collected by centrifugation, washed and dried in oven -> weighed. Insensitive (>10^9 cells/ml required), very slow (1 or 2 days in oven). Used for studies on fungi & filamentous bacteria
Wet weight determination: weigh pellet after centrifugation -> result usually less consistent than dry weight, insensitive (varies) but rapid
Direct (or total) cell counts
Example: microscope counts
Not very sensitive (> 10^7 cells/ml required)
Rapid method (completed in ~30 mins)
Uses counting chamber: side view of chamber showing coverglass and space beneath holds bacterial suspension. Top view: grid in centre of slide. In grid, bacteria in several squares are counted -> used to calculate conc of cells in original sample
Viable cell counts: serial dilution and plating (direct)
Plates with 30-300 colonies are counted
0.1ml aliquots are taken and spread onto surface of agar plates using sterile glass or plastic spreader
Viable cell counts: conc/filtration (direct)
Membrane filter on filter support-> water sample filtered through membrane filter (0.45um pore size will trap most bacteria) -> membrane filter removed and placed in plate containing appropriate medium -> incubation for 24 hrs -> typical colonies
Membranes with different pore sizes trap different microorganisms. Incubation times for membranes also vary with medium and microorganism
Most probable number (MPN) (direct)
Statistical estimate of probable pop in liquid by diluting and determining endpoints for microbial growth
One cell would give rise to turbid broth after O/N incubation
Used in water analysis
Turbidity (indirect growth measures)
Bacterial cells affect passage of light through a solution
Measured by light absorbance or its inverse, light transmittance
Very rapid method and often used
Inaccurate when cell densities are high
Measuring turbidity
Spectrophotometer has 2 scales: bottom scale shows absorbance and top scale: percent transmittance
Absorbance increases as % transmittance decreases so absorbance=1 when %transmittance=0
Determination of microbial number by measurement of light absorption. As pop and turbidity increase, more light is scattered and so absorbance reading increases
Desirable to maintain cells in exponential growth: applications of knowledge of bacterial growth
Useful in genetic and physiological experiments: cells in identical growth state, relative synthetic rates constant for all cell constituents thus reproducibility in repeated experiments
Useful in industrial fermentations to make primary metabolites (produced during active growth)
Applications of knowledge of bacterial growth: stationary phase is needed for other purposes
Secondary metabolites: products of metabolism synthesised after growth has been completed
E.g. antibiotics produced in stationary and death phases
Applications of knowledge of bacterial growth
Chemostat enables cells to be kept in exponential phase, at specific growth rate (provide constant supply of one essential nutrient at limiting conc = continuous culture system)
