15 - Bacterial growth and measurement of growth Flashcards
Generation time (g)
Time for a cell to produce 2 cells or for the population to double in number
Septum
forms down the middle of the cell, separating it into two during binary fission
Why does bacterial growth stop
- nutrients are used up
- metabolic wastes that may be toxic accumulate
- living space becomes limited
- aerobes may suffer from oxygen depletion
Significance of bacterial growth
- Human and animal health implications
- Environmental impacts (blooms)
- Industrial applications (medicine, cheese)
Stationary cells
Viable but non dividing and/or non viable cells
5 phases of bacterial growth in closed system
- Lag phase
- Exponential or log phase
- Stationary phase
- Death phase
- Long term stationary phase
Lag phase
- Cells have to adapt to new physiological conditions.
- Synthesis of cellular constituents (especially ribosomes and enzymes)
begins - Cell volume increases but no cell division
- Growth rate (k) and generation time (g) = 0
- Different genes are upregulated at different times to make needed components
- cell division and population growth begin at end of lag phase
Exponential (log) phase
- Bacteria grow and divide at max rate possible
- Population most uniform in terms of physiology
- k and g are constant
- Continues until limiting factors or toxicity drive culture into stationary phase
growth rate calculation
k = 1 / g (generations/hour)
if g = 20 mins (0.333 hr)
k = 1/0.333 = 3 gens/hr
Stationary phase of growth
- k = 0
- Cells not dividing because nutrients have been used up, or a toxin has built up and cell death balances cell division
- reached at `10^9 cells/mL
Properties of cells in stationary phase
- Tend to be smaller than exponential phase
- Different composition of cellular constituents
- available energy is used for maintenance (endospore formation)
Death phase
- Cells begin to die as nutrient deprivation, or toxic, waste buildup causes damage to cells
- k becomes negative
- Death rate usually logarithmic
- Some cells lyse, releasing nutrients to other bacteria
Long term stationary phase
- Can last months or years
- Population continually evolves. (actively growing cells use nutrients released by dying cells to tolerate toxins)
- Successive waves of genetically distinct variants
biofilms
Aggregations of microbes in complex communities, growing on surfaces and held together by extracellular polymers.
3 different methods of measuring bacterial growth
- Measure cell biomass
- Direct measurement of cell number
- Viable counting methods
3 types of cell biomass methods
- Dry weight determination
- Wet weight determination
- Spectrophotometry
Dry weight determination
- Cells in broth collected by centrifugation, washed, dried and weighed
- Insensitive and very slow
- Used on fungi and filamentous bacteria
Wet weight determination
- Weigh pellet after centrifugation
- Result less consistent than dry weight
- Insensitive but rapid
Spectrophotometry
- Bacterial cells scatter light which is proportional to biomass of cells
- Increases in cell concentration result in greater turbidity (cloudiness) and less light is transmitted.
- As turbidity increases, absorbance increases
- Sensitive and very rapid
- Inaccurate when cell densities are high unless diluted.
3 types of direct measurement methods
- Direct cell counts using counting chamber
- Staining with fluorescent stains
- Flow cytometry
Direct cell counts using counting chamber
- Slide creates chamber of known volume
- Cells on grid are counted, cells/mL calculated
- Not very sensitive, rapid
- isn’t affected by bacteria absorbance abilities
Staining with fluorescent stains
- Sample filtered and bacteria trapped on membrane
- Stained with nucleic acid fluorescent stains and counted under microscope
Flow cytometry
- Provides cell count plus detailed info of organism
- Stream of cells passed through beam of laser light
- Measures the amount of light that gets through and the amount scattered
- sensitive, rapid
3 types of viable counting methods
- Viable counts/standard plate counts
- Membrane filtration
- Most probable number (MPN)
Viable counts/standard plate counts
- Suspensions are diluted (1ml of previous dilution added to 9mL of water)
- Original sample, 10^-1, 10^-2, 10^-3, etc
- pour plate and spread plate method
- Single colonies then counted after incubation
- counts 30-300 are valid
- Sensitive but slow
Membrane filtration method
- Membranes with different pore sizes used to trap microorganisms
- Contain grids so colonies can be counted
- Allows bacteria present in low concentrations of water
- Sensitive but slow
Most probable number
- Serial dilutions used
- Tubes examined for turbidity
- One cell would give rise to turbid tube
- If no growth, no cells
- Table provides most probable cells/mL in og
- Useful where microbe cannot be cultured
- Very sensitive, slow
Primary metabolites
Produced during active exponential growth (enzymes, amino acids)
Secondary metabolites
Compounds not needed for normal cell growth but beneficial. Synthesised in stationary phase
Chemostat
Enables cells to be kept in exponential phase at specific growth rate by providing constant supply of one essential nutrient at a limiting concentration
