Chapter 7 Flashcards
How are different types of media classified?
3 bases:
Chemical composition: Defined (synthetic), complex
Physical nature: Liquid, semisolid, solid
Function: Supportive (general purpose), enriched, selective, differential
Explain how a biofilm is formed (steps)
Step 1: Ambient molecules precondition surface.
Step 2: Cell deposition (reversible).
Step 3: Cell absorption.
Step 4: [Some] cell desorption.
Step 5: Cell-cell signalling, onset of exopolymer production (cells become irreversibly attached to surface).
Step 6: Convective + diffusive transport of O2 and nutrients.
Step 7: Replication and growth.
Step 8: Secretion of polysaccharide matrix.
Step 9: Detachment, erosion and sloughing.
What is quorum sensing? How does it work?
Cell-cell communication within biofilms mediated by small proteins, “autoinducers”.
Autoinducers (ex. Acylhomoserine lactone (AHL)) released by cells and accumulate in concentration as microbes replicate, which also accumulate in a biofilm. As external concentration increases, it re-enters the cells by a diffusion gradient, and once inside at a threshold concentration, it induces the expression of target genes.
Name the phases of growth that occur in a BATCH culture. Describe what is happening in each.
- Lag phase: cell synthesizing new components to replenish spent materials (ATP, ribosomes, etc) or to adapt to a new medium/other conditions. (Length of lag phase depends on how closely batch reactor compares to cell’s natural environment).
- Exp (log) phase: rate of growth and division is constant and maximal = balanced growth (population most uniform in chemical and physical properties)
- Stationary phase: total # viable cells remains constant; metabolically active cells may stop reproducing; reproductive rate may be balanced by death rate.
- Death phase: 1) cells viable but not culturable (VBNC - dormant), 2) genetic programmed cell death
What are some of the causes of the stationary phase in the growth of a BATCH culture?
- Nutrient limitation
- Reproductive rate may be balanced by death rate (critical population level is reached)
- Toxic waste accumulation
- Limited oxygen or other terminal electron acceptor
What are some survival strategies adopted by cells in the stationary phase of the growth of a BATCH culture?
- Some starved cells decrease in size and are more difficult to kill as a result
- Some cells form endospores (for preservation)
Define the generation time (or doubling time).
Doubling time (td) or mean generation time (g) is the time for a population to double.
Mathematical expression:
td = g = (ln2)/mu = ~0.693/mu
Define specific growth rate.
Specific (mean) growth rate is defined using the following expression:
mu = (1/X)(dX/dt)
Where:
X = cell mass concentration (g/L)
t = time (h)
mu = specific growth rate (h^-1)
What is chemostat culture? How is it different from a batch reactor?
A chemostat is an OPEN system in which all chemicals (nutrients, biomass, etc) levels are CONSTANT at STEADY STATE. The rate of incoming medium equals the rate of removal of medium from the vessel with an essential nutrient in limiting quantity.
A batch reactor is a CLOSED system with nothing entering and nothing leaving the reactor. The reactor concentrations change with time.
Name all DIRECT ways of measuring cell growth and all VIABLE ways of measuring cell growth.
Direct:
- Counting chambers
- Electronic counters
Viable:
- Plating methods
- Membrane filters + staining
- Membrane filtration methods
How does the Petroff-Hausser counting chamber work in measuring microbial growth?
- Cells dyed to distinguish between live/dead cells
- Cells are measured as # bacteria/unit volume
- The counting chamber (grid) is of known volume
- Count # of cells in a section of boxes and multiple accordingly to approximate
- 25 squares, each 1mm^2 and depth 0.2mm
- Not much space for turbulence, relatively flat throughout so cells are equally distributed
- Easy to access, can stain live/dead cells
- Time-consuming (slow throughout since # sample runs/min)
How does an electronic counter work in measuring microbial growth?
- Cells forced through small orifice
- Movement of cells through orifice affects electric current
- Disruption of current counted electronically
- Easy and fast to process
- Requires a larger sample, difficult to distinguish between live and dead cells
- ALL particles w/ threshold voltage are counted (dust, debris, other particles)
How are spread-and-pour plate techniques performed?
- A diluted sample of cells is spread over solid agar surface or mixed with agar and poured into a Petri plate.
- After incubation, the number of organisms are determined by counting the number of colonies multiplied by the dilution factor.
- Results expressed as colony-forming units (CFUs)
Provide an example of a SPREAD plate technique.
Step 1: Small amount of sample pipetted into centre of solid medium.
Step 2: Glass spreader is sterilized by dipping into ethanol and briefly flaming.
Step 3: Spreader is cooled and used to spread sample evenly across plate.
Provide an example of a POUR plate technique.
The original sample containing cells is diluted several times (as performed in Lab 1, 3 and 4). Some of the dilutions are often mixed with liquid agar and poured into plates. Isolated colonies grow on the surface of the agar (appear round) or within the medium (appear lens-shaped).
