why is studying growth important
- control of microbial growth is desirable in many settings
- knowing growth or kinetic parameters or organisms can allow us to understand their physiology and manage them better
what is growth
- increase in cell size
- increase in cell number
what are the different phases of microbial growth and explain
lag phase = preparation phase, cells not dividing, adapting to environment and new conditions (can be short or long)
exponential (log) phase = maximal cell growth rate, cells are dividing, growing rapidly at their fastest rate, cells are most uniform and similar to each other
stationary phase = cell growth levels off as nutrients are depleted and waster products accumulate, leveling off, nutrients are used up
-> death rate = growth rate
death phase = insufficient nutrients leads to death rate>growth rate
-> not enough nutrients (depleted even further)
-> decrease in cell number
explain extended stationary phase
- if microbes can utilize microbial decay products as an energy resource, an extended stationary phase may be observed after initial death phase
- cells capable of using cell decay as food
explain exponential growth in more detail
- each cell divides at constant intervals
so the population is doubling at these regular intervals = generation time or doubling time
-> most uniform and consistent
-cell yield (number of cells/volume) is determined by nutrient availability - nutrient can limit growth in exponential phase
-> if all nutrients are present in excess, cell yield and growth rate level off at maximums that are limited by the rate of cellular processes (enzyme kinetics and transport processes) - these are the growth rate yield Y(g cells/ g substrate)
- maximum specific growth rate uMax (t-1) = max rate at which organism can grow
compare batch and continuous systems
batch -> nutrients are finite, concentration decreases over time, waste products accumulate
-> microbial growth will follow the lag, log, stationary, death pattern
continuous-> nutrients are continuously replenished, waste products and cells are removed
-> microbial growth can continue to proceed exponentially
-> no stationary phase reached because you keep adding nutrients
which organisms benefit from batch/continuous culture conditions
- gut bacteria (batch as well)
- slow growing bacteria
what is dilution rate
- D
- medium is fed at a constant rate, rate at which you are adding and removing media
- D=f/V
f= flow rate
v= reactor volume - effluent (which contains cells and medium) is removed at the same rate
explain growth in a chemostat
- fresh medium -> microbes grow in culture vessel -> waste bottle
- once the system reaches steady state the growth rate and the number of cells in the chemostat is constant
explain growth in a chemostat effect of D
- the growth rate in a chemostat at steady state is controlled by and is equal to the dilution rate
- once the system reaches steady state the growth rate and number of cells in the chemostat is constant
- change in biomass (W) over time in chemostat: dW/dt = uW-DW (growth rate biomass) - dilution rate biomass
-> W= constant, so u=D (growth rate=dilution rate)
what happens to the growth rate as D is increased
- increases
- the growth rate continues to increase as nutrients are added more quickly but eventually the cell number decreases as the cells cannot grow faster than they are removed = washout
- this occurs above uMax -> the maximum specific growth rate of the species you are working with
explain growth in a chemostat effect of Cr
Cr= the concentration of the limiting nutrient in the reservoir is another factor that affects microbial growth in a chemostat
- steady state is always attained but the number of cells at steady state is higher with higher Cr
- control the # of cells that exist in the chemostate
what is carrying capacity
- carrying capacity = the number of cells that can be sustained by the resources provided
what are 4 ways to measure microbial growth
- direct counts
- culture based methods
- biomass based methods
- molecular methods
what are direct counts
- cell counting chamber with microscope (must be relatively large population and evenly distributed)
- flow cytometry
-> narrow channels allows one cell to pass through laser beam at a time (must be well dispersed, not sticking together)
-> cells may be living or dead
-> live/dead fluorescent staining
-> other staining techniques can be used to differentiate cell types/ species
what are culture based methods
- viable counts
- spread plate and pour plate -> dilute sample, inoculate plates, count colonies
- filter plate -> filter is used to inoculate plate, count colonies
- small amount of sample is pipetted to the center of a solidified medium
- glass spreader is sterilized by dipping into ethanol and brief flaming
- spreader is cooled and then used to spread the sample evenly over the surface of the medium
what is the MPN method
- a dilution series of the sample is made, usually ten-fold dilutions are used
- 1.0ml of each dilution is used to inoculate triplicate tubes of growth medium
- the tubes are evaluated for growth, the first set of dilutions that fail to show growth is used to bracket a set of three dilutions
- the MPN is determined by consulting a table that has been established using statistical analysis
what is biomass quantification
- dry weight = requires a lot of biomass
- volatile and total suspended solids (VSS/TSS)= filter sample through glass fibre filter, dry at 105C -> change in mass of filter is TSS, then bake at 550C, 30 mins -> loss is the VSS (=biomass)
- spectrophotometry -OD600
- protein/chlorophyll = quantify the amount of these and convert to cell number based on a standard amount per cell
- all these methods require some kind of standard values or curve
what are molecular methods
- quantitative PCR (qPCR, aka real time PCR)
- single copy genes are present in most organisms and in only one copy
- includes genes for functions that are shared amongst all bacteria
- for this reason the sequences also tend to be evolutionarily conserved (ribosomal proteins, tRNA synthetases)
- quantify the number of template after each cycle using a fluorophore that binds to dsDNA and fluoresces
