Topic 4-L2 - Culturing Microbes Flashcards
Half of that weight of a microbial cell is
protein, ~ 25% is nucleic acid (mostly
ribosomes!), much of the rest is the cell envelope
Microbes also require lower
abundance molecules (e.g. enzymatic cofactors)
Although a lower % of cell material,
availability of these low-abundance
nutrients can dictate
growth of a microbe
CHONPS
Key elements to build core macromolecules by cells (protein, DNA)
Mg2+ plays key roles in stabilizing
negative charges in membranes, nucleic acids – also used by enzymes
Many organic “micronutrients” & trace metals are required a very low
amounts
Microbes that require these (often scarce) nutrients often evolve highly
efficient mechanisms for their uptake
Essential cations and anions for cells
Na, Mg,K,Ca,Cl
Growth media or culture media (medium, singular) can be highly variable depending on the
microbe…and for a given microbe
Defined media:
Media prepared by adding precise/known quantities of chemicals to water. Know the exact composition.
Complex media:
Contain extracts or digested organic material with an unknown composition. E.g. yeast extract, casein (milk protein) digests
Defined medium has the advantage that
you know what you’re
working with.
Complex media advantage is that they are very
common – cheaper, easier, work for a broad(er) array of different microbes
Other microbes can require a great number of growth factors – things like vitamins, amino acids, purines/pyrimidines, etc.
- Many organisms have an obligate symbiotic lifestyle
- Organisms that live in nutrient-rich environments (such as many lactic
acid bacteria)
Some microbes can make most or all of the organic molecules they need
- Many microbes that live in nutrient-poor environments
- Certain “flexible” microbes that adapt to many different environments
such as E. coli
Auxotrophy –
inability to produce a molecule you need for your growth. (Prototrohpy is opposite - can produce that molecule)
E.coli can grow in a complex media contains
Peptone and yeast extract
Selective media:
Used to isolate a limited range of microbes (often bacteria) – this could be a single species. Often a combination of positive (nutrients few organisms can grow on) and negative (substances that kill most microbes) selection.
Differential media:
Contain some sort of an indicator (e.g. a dye that changes colour) when particular organisms are present
Enrichment culture:
Similar idea to selective media…but less selective and richer medium. Promotes growth – increase numbers from isolates to make it easier to isolate a particular microbe. Usually somewhat selective.
Solid plates useful for
isolating single colonies –(ideally) originates from a single cell that grows to large numbers & can be readily seen
- identify morphology, contamination, start pure cultures
Liquid cultures (e.g. used in experiments) should generally be started using
isolated single cultures
streaked on agar plates
All media is selective?
Yes
The great plate count anomaly
When “natural” samples are plated on generic growth plates (meant to be “non-selective”)…99.9% of microbes don’t grow.
We cannot culture the vast majority of microbes? Why or why not
NO we cant, cuz of syntrophy
syntrophy
(microbes feeding off one
another – microbes require other community members to grow)
Counting microbial cell numbers:
- Direct count: counted using a microscope
- Viable plate counts: colonies counted
- Turbidimetric: Absorbance of light by a microbe growing in a uliquid culture is measured in a spectrophotometer
- Other indirect methods: O2 consumption, CO2 production,metabolic activity. Quantitative PCR to determine the number of
“genome equivalents”.
Direct – microscopic counts
Known volume, gridded microscope.
- doesn’t require growth (advantage)
- prone to inaccuracies, requires staining. Can confuse microbe with debris.
Viable plate counts
- reliable and common
- need to known how to grow microbe
- assumes colonies emerge from single cell. (Problem when clumping/aggregating)
- assume ALL cells grow to form colonies (some cells are viable but aren’t culturable)
Turbidity measurements
- microbes scatter light, amount of light scattered proportional to liquid sample. (Common and reliable)
- correlation b/w optical density and cell #
- only for single isolated pure cultures in liquid sample
A significant advantage of turbidity measurements is that they are not
labor intensive and growth can be continuously or regularly measured over time
Disadvantage for turbidity measurements
- Limited useful range:
At low low cell numbers, insufficient light scattering not detected. High enough cell numbers saturate signal (no longer linear)
Microbial “growth”
refers to increase in population size – cell division resulting in multiplying in numbers
Generation time (doubling time):
The amount of time it takes how long it takes for one cell to become two (cells to double in numbers & mass). Varies greatly depending on microbe & growth conditions
Batch cultures -
cultures in a fixed volume in a closed container like a flask or a test tube.
Continuous cultures –
cultures within systems where waste product are being removed and new media fed in
Growth phases of batch culture
- Lag phase : adjustment phase
- exponential phase
- stationary phase: food limited, waste accum.
- decline phase
Cell numbers double at some regular interval – this interval can vary greatly depending on
microbe/growth conditions
Generation time formula
Generation time = (growth time) / (number of generations)
- g = t/n
Growth during exponential phase
Total number of cells at any time (N t) will be equal to the starting number of cells (N0) multiplied by 2n, where n is the number of generations
- Nt = N0 x 2n
Exponential growth can lead to very large numbers of microbes accumulating in a short time
In the real world, cells
rarely grow exponentially at such as fast rate…but it can happen.
Continuous culture
Cell can be grown indefinitely in the lab if we can remove waste products (and cells) and add fresh media
Chemostat (continuous culture device) can be used to
grow cells at a steady state – culture volume, number of cells, nutrient-waste status all kept constant.
- Cells grow at same rate that they are removed
- useful for industrial/experimental applications
Dilution rate -
volume always added & removed at same rate - controls growth rate in cont. culture
- (To maintain same number of cells…if you replace the full volume every 2 hours, cells must double every 2 hours)
Nutrient availability in the medium controls how
many cells you’ll have (density of culture)
More nutrients – equilibrium will be established with more cells/ml – greater yield
planktonic growth
(free-living organisms in liquid)
sessile growth
(growth attached to a surface)
Growth on surface can develop into
biofilms –
cells encased in polysaccharide matrix attached to surface
In many cases, bio films are complex
communities with significant
differentiation (cells with different
properties in different layers of biofilm)
Biofilm formation can start with a
planktonic cells attaching to a surface
via appendages such as pili, fimbriae or even the flagellum
After attachment to surface, the next steps for bio films formation is
- colonization
- development
- disperse
Colonization begins and the cells
grow (multiply) and produce extracellular polysaccharides
During development, cells change their biological program - express unique
combinations/amounts of genes to facilitate this biofilm lifestyle. Different cells (e.g. surface exposed vs. lower layers), different programs.
disperse –
resume planktonic state. Form a new biofilm elsewhere?
