Flashcards in Microbial Lifestyles and Metabolism (Dr. Kreft): How Microbes Grow Deck (22):
Biomass and cell number can be used as measurements of?
Explain different ways biomass can be a measurement of growth!
- Protein assay ( 50% biomass is protein and does not change much)
- Dry mass (dry washed cells and weigh them)
- Optical density (OD)
L> particles scatter light, bacteria are particles. OD is the measure of biomass rather than number of cells. Measure of cell number only so far as numbers correlate with mass (balanced growth)
Explain different ways cell number can be a measurement of growth!
- total count: count cells in known volume under the microscope (counting chamber) -> cells/mL
- Viable count - CFU (colony forming units) - spread 0.1mL of (diluted) culture on agar plate and count # of colonies forming ...CFU/mL
Of the various ways to measure growth what is the most convenient method?
- Optical Density (OD)
- If OD is too high (>0.5) have to dilute sample as response is no longer linear
- OD proportional to biomass per volume
- OD not proportional to cell number per volume
- Cell number correlates with biomass only if the cell size is constant i.e. in balanced growth but not in stationary phase
What are the two types of systems you can have for growing microbes?
- open system
-closed system - no addition of substrates during growth or can be closed off to exposure to environment
What are the four growth phases in batch culture?
Explain the lag growth phase!
- conditions change from pure culture to new one. Microbes respond to this change - takes time to adapt therefore growth rate does not start strong right away. Gene expression changes. Specific growth rate will not increase until exp phase.
Explain the exponential growth phase!
- composition of cells does not change. Just growth. High cell density increase. Substrate can run out quickly form high to low. Examine oxygen consumption here?
Explain the Stationary growth phase!
- oxygen is used up quickly etc...enter a shock since substrate is gone. Gene expression changes to adapt. Use up internal resources
Explain the death growth phase!
- death is exponential after some transition. All internal resources are used up
Growth yield =??
- biomass formed per substrate consumed
Is a continuous culture (chemostat) an open or closed system?
- Open: resources enter and biomass and left over resources leave
- structure: sterile air or other gas + fresh medium from reservoir via flow rate regulator----> culture
L> gaseous headspace + culture vessel
L> waste products etc leave via overflow and effluent containing microbial cells as well.
- microfluidics allows the study of single cells growing in a chemostat
- if state of the system does not change with time (all variables are constant)
**low dilution rate (D) (20% of maximal specific growth rate)
*** increase= decrease
*** specific growth rate = dilution rate u(S) = D
L> obtained at a certain substrate concentration S
Steady state : high dilution rate? what happens with biomass and substrate over time?
- high D (80% of maximal specific growth rate)
- biomass increases and plateaus
- substrate gradually decreases...as it falls biomass plateaus
After running many steady states at different D we can plot these steady states as a function of?
- dilution rate
- it is the parameter we can control
What happens to the graph when you plot specific growth rate versus substrate concentration? (steady states)
- Monod kinetics (michaelis menten enzyme kinetics)
- we control D forcing the bacteria to grow at that specific growth rate
- substrate affinity, the substrate concentration at which half Umax is reached....the lower the Ks, the higher the affinity and the steeper the increase
Why should we use a chemostat?
- can keep many cells under constant conditions - ideal for systems biology and physiology
- can study dependence of growth on substrate concentration (monod kinetics, yield not quite constant due to maintenance energy, cells growing at different rates differ in ribosome content, gene regulation)
Is the chemostat more like the natural environment vs batch culture?
-open system reaches steady state unless perturbed, slow growth rates in chemostats and nature, low substrate consecration in chemostat and nature, can enrich bacteria from nature which have high substrate affinity
What are two huge areas of study, chemostats can be used for?
- Evolution: experimental evolution in the lab
- Ecology: competitive exclusion principle