6-2: Culturing microbes Flashcards
What is most of a microbial cell weight made of?
50% Proteins, 25% nucleic acid, rest is the cell envelope
What are the key elements required to build the core macromolecules used by the cell
C, H, O, N, P, S
Where do H, O, P, S come from?
Organic molecules. HO can come from water, most cells can assimilate inorganic PS.
What does Mg2+ do
Stabilizes negative charges in membranes, nucleic acids
What is Fe required for?
Needs to act as a cofactor for specific enzymes
What is growth media/culture media made of?
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 (eg. yeast extract, casein)
What are advantages of defined media vs ccomplex media?
Defined = know what you’re working with
Complex = common, cheaper, easier, works for a broader number of microbes
What types of microbes can make most organic molecules?
Many microbes that live in nutrient poor environments and can adapt to different environments eg. E. coli
What types of microbes require lots of growth factors?
Obligate symbiotes and organisms that live in nutrient rich environments (eg. lactic acid bacteria)
What is auxotrophy? What is prototrophy?
Cannot produce a molecule needed for growth.
Phototrophy = can grow molecules needed for growth
What is selective media
Used to isolate limited assortment of microbes, maybe a single species. Uses a combination of 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 (eg. environment). Somewhat selective
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 “Great Plate Count Anomaly”
What is syntropy
Microbes feeding off one another, microbes require other community members to grow. Prevents us from culturing the majority of microbes in isolation
What are the four methods of counting microbial cell numbers?
- Direct count - count cells using microscope
- Viable plate counts - small sample spread on agar plates then colonies are counted
- Turbidimetric - absorbance of light measured by spectrophotometer
- Indirect methods - O2 consumption, CO2 production, quantitative PCR
How do direct microscopic counts work? Disadvantage?
Known volume added to gridded microscope slide, cells counted under microscope
No growth required (advantageous), but very laborious /inaccuracies (viable vs dead, needs staining)
How does viable plate count work? Disadvantage?
Uses serial dilutions
Need to know how to grow specific microbe
Assume colony from single cell (clumping/aggregating problem)
Assumes that all cells can form colonies (some cells are non-culturable)
How does turbidity measurement work?
Microbes scatter light proportional to number of microbes in sample
Very reliable and commonly used
What does some requirements for turbidity measurements?
Only works in pure cultures and liquid cultures
Requires you to determine the occrelation between optical density at chosen wavelengths and cell numbers
What is a bid advantage of turbidity measurements?
Not labour intensive, can be continuously measured over time
Major disadvantage of turbidity measurement?
Limited range
Low cell # = insufficient light scattering
High cell # = signal saturated
How are turbidity measurements typically plotted?
TIme on X axis, OD on Y axis (sometimes on a log scale)
What is microbial growth
Increase in population size via cell division (increase in number of cells)
What is generation time
Amount of time it takes for one cell to become two cells. Depends on microbe/growth conditions
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 time)
What factors increase the lag phase?
Different environment, metabolically silent cells
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
What is the equation for generation time?
Growth time / number of generations
g = t/n
What is the equation for exponential phase growth?
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
What is the difference bewteen planktonic vs sessile growth?
Planktonic = free living organism in liquid
Sessile = growth attached to surface (common in real world)
What can sessile growth can develop into?
Biofilms - cells encased in polysaccharide matrix, with significant cellular differentiation
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
