Cultivation independent methods - DNA from unculturable bacteria can be amplified and sequenced by PCR
Sequences can be used to produce fluorescent probes that will bind to complementary DNA (Fluorescent In Situ Hybridization FISH)
FISH - Cells fixed in place
Fluorescently labeled probes consi...
MCB 2610 Exam 2 Questions and
solutions
Cultivation independent methods - DNA from unculturable bacteria can be
amplified and sequenced by PCR
Sequences can be used to produce fluorescent probes that will bind to
complementary DNA (Fluorescent In Situ Hybridization FISH)
FISH - Cells fixed in place
Fluorescently labeled probes consisting of 165 rRNA gene sequence sent to attach
to RNA
View in Epifluorescence microscope
Metagenomics - DNA is isolated from an environmental sample and sequenced
Metagenomic information must still be confirmed in cultured organisms
Microbial consortia - Microbes that can't grow on their own
Direct counts - Load known volume into gridded slide
Count under a light microscope
Flow Cytometry - Microbial suspension forced through small orifice with a laser
beam
Movement of microbe through orifice impacts electric current that flows through
orifice
Instances of disruption of current are counted
Specific antibodies can be used to determine size and internal complexity
,Spread plate technique - used to get a living (viable) cell count
Small amount of sample is pipetted onto a solid medium then spread around that
medium
Measured in CFU colony forming units
Pour Plate - Original sample is diluted multiple times and the most dilute ones are
poured into a plate with liquid agar
Isolated cells grow into colonies on the surface and inside of the medium
Measured in CFU colony forming units
Calculating CFU/mL - Average cell number / (dilution X volume plated)
What if cells are already very diluted? - A filter apparatus can concentrate cells
Sample is filtered through a membrane filter then the contents that don't go through
are transferred to a medium and incubated
Turbidity - Spetrophotometer sends light through a liquid culture
If the tube is cloudy, light wont reach the other side because it will hit all the
bacteria in the tube
This can give a rough estimate of cell density in the tube
Higher absorbance = higher population in culture
Indirect measurements of cell mass - Dry weight - time consuming and not very
sensitive
Quantity of particular cell constituent - DNA protein ATP
, Growth - Population growth vs growth of actual cells
Growth Curve - Observed when microorganisms are cultivated in batch culture
(closed culture)
Usually plotted as a logarithm of cell number vs. time
Has 4 distinct fases
Lag phase - Cell synthesizing new compounds
ex. to replenish spent materials, adapt to new conditions or medium
Exponential phase - Rate of growth and division is constant and maximal
Population is most uniform
Stationary phase - Closed system
Population growth eventually ceases
Reasons - nutrient limitation, limited oxygen availability, toxic waste accumulation
- can make environment too acidic
Stationary phase and starvation response - Activate survival strategies:
Morphological changes - endospore formation, decrease in size
RpoS protein assists RNA polymerase in transcribing genes for starvation proteins
Senescence and Death Phase - Number of viable cells declines exponentially
Two alternative hypotheses:
Cells are viable but not culturable (VBNC) - cells alive, but dormant, capable of
new growth when conditions are right (programmed cell sterility)
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