6-2: Culturing microbes Flashcards
Most of a microbial cell weight made of
Proteins, then nucleic acid
What are the key elements required to build the core macromolecules used by the cell
C, H, O, N, P, S
Where do H and O come from
water
What does Mg2+ do
Stabilizes negative charges in membranes, nucleic acids
Where do microbes get H/O/P/S
organic molecules
What is growth media/culture media
Highly variable depending on the microbe
What is defined media
Media prepared by adding precise/known quantities of chemicals to water (exact composition known)
What is complex media
Contains extracts or digested organic material with an unknown composition
Advantage of defined media? Complex media?
Defined = know what you’re working with
Complex = common, cheaper, easier, broader
The inability to produce a molecule you need for growth is… The opposite is…
Auxotrophy = cannot
Phototrophy = can
Example of a microbe that can make all/most of the organic molecules they need from a few basics, lives in nutrient-poor environment
E coli
Microbes may require a great number of growth factors, like…
Vitamins, a.a., purines/pyrimidines
What is selective media
Used to isolate limited assortment of microbes, maybe a single species. Positive selection (nutrients few organisms can use to grow) and negative selection (kill most microbes)
What is differential media
Contain some sort of an indicator (e.g. dye) when particular organisms are present
What are enrichment cultures
Similar to selective, but less selective and richer. Promotes growth of particular microbes from samples
What are solid plates good for?
Solid = isolate single colonies, originate from single cell, easily seen, pure cultures
How are all media selective?
99.9% of microbes in a sample don’t grow on plates - most microbes not culturable
What is syntropy
Microbes feeding off one another, microbes require other community members to grow
Four methods of counting microbial cell numbers
- Direct count - count cells using microscope
- Viable plate counts - small sample spread on agar plates then counted
- Turbidimetric - absorbance of light measured by spectrophotometer
- Indirect methods - O2 consumption, CO2 production, etc
How does direct count work? Disadvantage?
Volume added to gridded microscope slide, cells counted under microscope
No growth required, but very laborious /inaccuracies (viable vs dead)
How does viable plate count work? Disadvantage?
serial dilutions
Need to know how to grow specific microbe
Assume colony from single cell
Some cells viable but non-culturable
How does turbidity measurement work?
Microbes scatter light proportional to number of microbes in sample
Reliable, but only works in pure cultures and liquid cultures
Not labor intensive, growth continuously measured
Major disadvantage of turbidity measurement?
Limited range
Low cell # = insufficient light scattering
High cell # = signal saturated
What is microbial growth
Increase in population size via cell division
What is generation time
Amount of time it takes for one cell to become two cells.
What are batch cultures
Cultures in a fixed volume in a closed container like a flask/test tube
What are continuous cultures
Cultures within systems where waste products are being removed and new media fed in
What is the lag phase in batch cultures
Period of slow/no growth as microbes adjust to new environment (variable)
What is the exponential phase of batch cultures
Growing population doubles at regular intervals. Nutrients still available, little waste
What is the stationary phase of batch cultures
Nutrients begin to be limiting, waste accumulation inhibits growth
Little to no net growth (but bacteria still growing and dying)
What is the decline phase of batch cultures
Cells start to die, net decline
What does generation time depend on? What is the equation
Depends on microbe/growth conditions
Generation time = (growth time) / (number of generations)
Exponential phase growth equation
Nt = N0 x 2^n
n=number of generations
Is long term exponential growth feasible?
No, would lead to large numbers of microbes accumulating in short time
Planktonic vs sessile growth
Planktonic = free living organism in liquid
Sessile = growth attached to surface (common in real world)
Sessile growth can develop into
Biofilms - cells encased in polysaccharide matrix
Biofilm formation process
Planktonic cells attach to surface via pili, fimbriae, flagellum
Colonization begins (cells multiply) and produce extracellular polysaccharides
Cells change biological program to facilitate biofilm lifestyle
Some cells begin to disperse, resume planktonic lifestyle
