Study Unit 4 Flashcards
What is cell growth?
Growth is a result of both cell division and a change in cell growth
Cell growth phases
- Lag phase
- Exponential phase
- Deceleration phase
- Stationary phase
- Death phase
When does the lag phase take place?
Directly after innoculation
What happens during the lag phase?
The cells adapt to their new environment by reorganizing their molecular constituents. Sometimes additional enzymes are synthesized and other times the enzymes are repressed
What happens during the exponential phase?
The cells multiple rapidly. Balanced growth occurs and the average composition of a single cell remains approx. constant.
Cell metabolism is set up to achieve maximum rates of production.
What happens during the deceleration growth phase?
Growth starts to decelerate due to the depletion of essential nutrients or the accumulation of toxic by-products.
The rapidly changing environment results in unbalanced growth where cell composition and cell size will change.
The stresses induced by nutrient depletion or toxic waste accumulation cause a restructuring of the cell to increase the prospects of cellular survival in a hostile environment.
What happens during the stationary phase?
The net growth rate = 0. Growth rate = death rate.
The cells are still metabolically active and produce secondary metabolites.
Cell lysis occurs and thus cryptic growth.
Cells don’t grow but they produce secondary metabolites.
Primary metabolites
Growth-related metabolites
Secondary metabolites
Nongrowth-related metabolites
What happens to cell number density and cell mass during the exponential phase?
Cell number density (number of cells) and cell mass increase exponentially with time.
What happens to the mass concentration during the stationary phase?
Remains constant
What happens to the number of viable (living) cells during the stationary phase?
Decrease
Cryptic growth
When cell lysis occurs the remaining viable cells use the lysis products as nutrients
What happens during the death phase?
Depletion of nutrients or toxic product accumulation
Is it easy to differentiate between stationary and death phase?
No
Why is the quantification of cell concentration important?
To determine the kinetics and stoichiometry of microbial growth
How can you determine cell concentration?
DIRECT METHODS
INDIRECT METHODS
Why are direct methods of determining cell concentration not always possible?
Suspended solids may be present as it is hard to differentiate between suspended solids and cells
Methods of determining cell number density
Petroff-Hausser method
Direct plate counts
Particle counter
Nephelometer
How is cell number density determined through the Petroff-Hausser method?
A callibrated grid is placed over the culture chamber. The number of cells per square grid is counted under a microscope.
Culture medium should be clear and free of particles that could hide cells or be confused for cells.
Hard to use this method for mould because moulds have a mycelial structure.
How do you ensure statistic reliability of the Petroff-Hausser method?
At least twenty blocks should be counted
How do you distinguish between live and dead cells in the Petroff-Hausser method?
Through the use of stains
How is cell number density determined through the plate counts method?
Culture samples are spread onto Agar surface on a petri dish, and the plates are incubated.
The number of colony forming units (CFUs) are determined from this.
Which cells are the plate count method more suitable for?
More suitable for bacteria and yeasts
Less suitable for moulds
How is cell number density determined through the particle counter method?
Cells have relatively high electrical resistances. An electrolyte solution containing the cells is placed inside the PC. A vacuum is applied over the inner tube which causes the cells to be sucked through the aperture hole.
The poor conducting cells alter the cross section of the conductive microchannel.
If these particles are less conductive than the surrounding liquid medium the electrical resistance across the channel increases causing the electrical current passing across the channel to briefly decrease.
By monitoring the pulses in electric current, the number of particles for a given volume of fluid can be counted.
What does the number of pulses in the current measure?
The number of particles
What does the amplitude of the pulses in the current indicate?
The size of the particles
Which organisms is the PC method not suitable for?
Mycelial organisms
How is cell number density determined through the nephelometer method?
Light enters through a slit and particles invisible to the naked eye scatters the incoming light.
Scattered light is measure by the phototube.
What is the turbidity of a liquid?
Haziness of a fluid caused by individual particles that are generally invisible to the naked eye (like smoke)
(transmitted light is measured)
What is the intensity of scattered light measured using a nephelometer proportional to?
The cell concentration
For what do nephelometers give the best results?
Dilute cell and particle suspensions
Methods of determining cell mass concentration
Determination of dry weight
Packed cell volume
Photospectrometry
How is cell mass concentration determined through the method of dry weight?
Samples of culture broth are centrifuged/filtered and then washed with a buffer solution or water.
The washed cell mass is dried at 80 degrees Celsius for 24h.
Then dry cell weight is measured.
When is the dry weight method applicable?
Only applicable for cells grown in a solid-free medium
If non-cellular solids like cellulose or molasses are present the dry mass will be inaccurate
How is cell mass concentration determined through the packed cell volume method?
Method in which cell concentration is rapidly but roughly determined.
Fermentation broth is centrifuged in a tapered graduate tube under standard conditions and the volume of the cells are measured.
How is cell mass concentration determined through the photospectrometry method?
A spectrometer is used to measure the turbidity (optical density) of the medium.
Light enters the slit and the particles in the sample scatter the light. Light that is not scattered is measured by the phototube.
Calibration curve is used to interpret measured results
(optical density vs. dry weight measurements (concentrations)).
If an unknown sample is now analyzed the absorbance is related to a certain sugar concentration on the calibration curve.
What is important to note when using photospectrometry to determine cell mass concentration?
Any other solids/light-absorbing should be absent
Wavelength is used that minimizes absorption by the medium components and regular blanking should be done.
How does protein, DNA and RNA play a role in measuring cell mass concentration?
Protein, DNA and RNA can be measured as indirect measures of CELL GROWTH. During a batch growth cycle the concentration of these intracellular components change with time.
What are the indirect methods of determining cell mass concentration?
Product formation
Substrate usage
pH changes
Viscosity changes
How is product formation used to determine cell mass concentration?
Products of cell metabolism can indicate cell growth.
How can changes in pH indicate cell mass concentration?
Indicates nutrient uptake and microbial growth (titration)
Nitrate in pH method of determining cell mass concentration
Nitrogen used during cell metabolism. H+ are removed from solution in proportion to the amount of nitrate that is taken up. The amount of acid added to then balance the pH is directly proportional to cell growth.
Nitrate in pH method of determining cell mass concentration
Nitrogen used during cell metabolism. H+ are removed from solution in proportion to the amount of nitrate that is taken up. The amount of acid added to then balance the pH is directly proportional to cell growth.
How can changes in viscosity indicate cell mass concentration?
Viscosity changes during some fermentation processes.
The change in viscosity will be directly proportional to cell metabolism.
Three major categories of microbial growth
Growth associated product formation
Non-growth associated product formation (specific production rate is constant)
Mixed growth associated product formation
What happens during growth associated product formation?
Products form simultaneously with the microorganisms
When does non-growth related product formation take place?
During the stationary phase when growth rate = 0
When does mixed growth associated product formation take place?
During the slow growth phase and the stationary phase
What does it mean if alpha is zero?
Product is only growth associated
Growing cells produce cells in constant proportion to their own growth
What does it mean if beta is zero?
Product is only non-growth associated
Product in direction proportion to cell concentration
What do we know about alpha and beta during mixed growth product formation?
They will be positive values
Net specific growth rate formula
μnet = (1/X) * (dX/dt) = μg - kd
μg: gross specific growth rate
kd: rate of loss of cell mass
What is growth yield?
Shows by how much cells will increase as the substrate is consumed
Which environmental conditions affect microbial growth and product formation?
Temp.
pH
Dissolved oxygen concentration
What effect does temp have on cell growth rate?
The cell growth rate approximately doubles with each 10 degrees Celsius increase.
If temperature is higher than optimal temperature, growth rate decreases and cells die more rapidly than they grow
Are optimal temperature for growth and optimal temperature for product formation the same?
No they may differ
What happens to pH if ammonia is the only nitrogen source?
H+ ions are released
pH decreases
What happens to pH if N2 is the only nitrogen source?
H+ is removed
pH increases
What effect does dissolved oxygen concentration have on cell growth rate?
Above critical oxygen concentration growth is independent of [O2]
