Quantifying Microbial Populations Flashcards
oligotrophic
- low levels of nutrients
(most natural enviro’s)
most microbes live in _____ states until …..
in growth arrested states (VBNC) until conditions are favourable to grow again
when there is no metabolic activity the cell is likely in the form of an _____
endospore
what is cell death
depends on the cell
-there are levels of deadness
sessile vs planktonic
what do sessile organisms tend to do?
sessile = found on surfaces
~ tend to cluster together into biofilms
planktonic = free floating
are biofilms difficult to remove?
YES, can have emergent properties like UV resistance
life within a biofilm (2)
- dynamic
- environment is very heterogeneous and then so are the members within
how can bacteria protect themselves in a dormant state
biofilms
what is quorum sensing
use signals to work together and turn on genes
ex. biofilms
bioluminescence
axenic cultures
pure cultures
methods of quantifying microbes
direct = microscopic counts
indirect = colony counts, turbidity measures
methods of quantifying viruses
direct = microscopic counts
indirect = plaque assays
what are microscopic cell counts
direct method
- dilution of cells is added to a special slide containing a grid
- sample must be stained with vital dye to determine live from dead
pros and cons of microscopic cell counts
pros - quick, inexpensive and easy
cons - not suitable for very small bacteria / very low densities
how can u increase or decrease the cell concentration for a microscopic count
decrease
- serial dilution
increase
- filter sample thru polycarbonate membrane
- the cells trapped on the filter and stained with fluorescent molecules
- count under an epiflourescence microscope
microscope vs colony counts
microscope - directly counting # of microbes seen
colony - you are counting colonies (not cells) so it’s indirect
describe colony counts
dilutions are added to a solid culture media and incubated
- only viable cells grow to form a colony !!
- not good for very low cell density
ideal #s for colony counts
30-300
when calculating you want the plating volume to be in ???
mL
how can u increase or decrease the cell concentration for a colony count
decrease
- serial dilution
increase
- use filtration and then place on agar plate
describe turbidity (absorbance) measurements
used to estimate the number of viable and non viable cells
- higher absorbance = higher cell concentration + higher turbidity
to estimate cell concentration from optical density you need a _____
standard curve
*both scales linear
how is most cell counting done nowadays
via qPCR or metagenomics
3 methods for culturing a virus
- embryonated egg culture
- cell culture
~ cells are grown in a dish and then the virus is added and multiple - live animal culture
how can we get a direct count of a virus
using a SEM
how can u indirectly count viruses
- lysis
- form viewing morphological changes
what are bacterial lawns
when an agar plate is totally covered in a thin layer of bacteria
how can you count viruses from a plaque assay
spread viruses over a bacterial lawn and then count the plaques (clear spots) that form
~ only viable, lytic phages will make plaques you can count
*measured in pfu / 1 plaque = 1 virus particle
