Lecture 9: Enumeration of Growth Flashcards
Be able to sketch and label a bacterial growth curve (x- and y-axis as well as phases).
X-axis is time and y-axis is log N.
Explain what happens in each phase of a bacterial growth curve.
Lag Phase: Bacteria adapting to new growth conditions.
Log Phase: Binary fission; all daughter cells survive (2-4-6-8-exponential increase)
Stationary Phase (mimics how the cells in our body work): Triggered when nutrients run out, there is too much waste, and cells run out of space. Cell continues to divide but only one daughter cell will survive (no change in number of cells). Persistor cells: Dormant cells that have a low metabolism (most metabolic processes turn off) in order to reduce the amount of energy and nutrients used up. Often antibiotic resistant. Common in biofilm environments. Not mutant, what is different is the way that they express their genes.
Decline Phase: Cells are still dividing but not very many of them. The number of cells dying is greater than dividing cells.
Be able to use the growth equation to model bacterial growth.
Nt = Noe^ut
- –Nt=final number of cells
- –No=principle number of cells
- –u=growth rate constant (per hour). If higher than the graph would have a steeper curve.
- –t=time
- –Generation time: Time it takes one cell to become two cells.
- applies only for log phase*
Explain the difference between primary and secondary metabolites, and give an example of each.
A primary metabolite is the growth/cell division happens. It contains macromolecules, growth factors, gas, acid. Occurs during exponential phase.
A secondary metabolite is cell survival. Contains endospores, antibioitics, biofilms (glycocalyces and pili), cells that sense crowding. Produced during stationary phase.
Explain why it takes a very long time for 100% of the bacteria in a culture to die.
Persistor cells are geared for survival. There are mixed cultures with subpopulations.
Cite 3 differences between a continuous culture and a batch culture.
Continuous cultures contain a reservoir (chemostat) that continuously administers nutrients while batch culture only gets nutrients once. Continuous cultures avoid stationary phases, it is constantly in log phase (growth equation cannot be used because it is a constant amount of cells). Continuous cultures are used to study primary metabolites and to change the growth rate. Ex. administer more nutrients= faster growth and vice versa.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Direct counts- Petroff-Hauser chamber
Counts all cells, dead and alive. Allows microscopic counts of all cells within a grid square of known size. Physically looking and counting so you can be sure that you are looking at a cell. Only for non-motile cells.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Direct counts- Coulter counter
Counts all cells, dead and alive. Measures interruption of electric current as cells pass through counter. You can count large volumes. Need a really clean medium because dirt will either clog up the pore or be counted as bacteria.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Viable (indirect) counts- Dilution series + pour plate
Counts alive cells only. If cells are too concentrated, then they must be diluted so that there aren’t a lot of cells on the plate (30-300 cells is considered countable). A fecal sample is very concentrated.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Viable (indirect) counts- Dilution series + spead plate
Counts alive cells only. If cells are too concentrated, then they must be diluted so that there aren’t a lot of cells on the plate (30-300 cells is considered countable). A fecal sample is very concentrated.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Viable (indirect) counts- Membrane filtration method
Only counts live cells. If the cells are too diluted then you must concentrate them before you dilute them. Measure drinking water would not be very concentrated. Holes are about 0.2um. The bacteria will collect on the filter and you just lay the filter on the plate and colonies can be formed via the nutrients from the plate. Would not work for milk or oatmeal samples.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Viable (indirect) counts- Most probable number method
Only counts live cells. Not actually directly counting. Assessing a given average, + or - indicate if there are cells in the upside tube. + means cells. - means no cells. Can assess the number of colonies in very dilute cultures.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Biomass measurements- turbidity
Counts both living and dead cells. Fast counting. More cells means more light scattered which means higher optical density (measured via spectrophotometer). If sample is too dilute, light will not be scattered. If material that the cells are in aren’t clear, it won’t work. Mixed cultures with cells of different shapes will scatter light differently.
Know how and when to quantitatively measure bacterial growth by each of the following methods: Biomass measurements- total cell mass
Counts both living and dead cells. Fast counting. Inaccurate because you can weigh things without cells, not all cells weigh the same, and you have to have about 10 million cells to have it weighted accurately. (Bacteria do not weigh a lot).
Know how bacterial growth can be quantified by measuring the accumulation of primary metabolites such as acid and gas, or by measuring ATP formation.
Gas: Measured in durham tube.
Acid: Measured via pH meter.
ATP: Luciferase lights up in the presence of ATP. Problem: Cells in different phases of the growth cycle produce different amounts of ATP. Assesses whether any cells are alive in the culture.
secondary metabolites not produced during cell growth and can therefore not be measured
