Lecture 4- Bacterial counts Flashcards
what is a continuous culture?
microbes in an open system with a fixed volume
what is the most common type of continuous culture device?
chemostat
what does a chemostat allow for? what is controlled?
growth rate of culture is controlled independently
population density of a culture is controlled independently
both occur simultaneously
what is a dilution rate?
rate at which fresh medium is pumped in and spent medium is pumped out
what controls population size and growth?
limiting nutrient
microbial counts can be counted using a petroff- hausser counting chamber, how does it work?
it has a grid and in each grid you count how many microorganisms there are
what are 8 limitations to microscopic cell counts?
- cant distinguish between live and dead cells without special stains
- small cells can be overlooked
- precision is difficult to achieve
- if stain is not used then need to use phase- contrast microscope
- low density cells are hard to count
- motile cells need to be immobilized
- debris in sample can be mistaken for cells
- cells may move, some form clumps
what is flow cytometry? what does it use?
its an alternative method that can be used to count the total number of cells that uses laser beams, fluorescent dyes, and electronics
what are viable cell counts?
counts that only measure living cells
what are two main ways to perform viable cell counts?
spread plate method
pour- plate method
what are the 3 issues with viable cell counts?
- requires lots of preparation and incubation time
- counts can be unreliable
- a single medium will never grow every microbe (some will thrive and some will die, they all have different growth requirements)
what is the great plate anomaly?
direct microscopic counts of viable (alive) cells reveal way more organisms then those on plates
why do microscopic counts give more counts of microbes?
- because they count dead cells
- different organisms may have different requirements for growth (we dont know all the requirements for all organisms)
what percentage of microbes are culturable?
1-10%
what is a spectrophotometric count example?
turbidity
how does turbidity spectrophotometry work?
light
light is absorbed
goes through curvette or tube
photocell detects how much comes through
spectrophotometer puts it in units of OD
what is turbidity spectrophotometry based on?
fact that bacteria behave like small particles (absorb and scatter light)
if theres a large number of particles what does that mean for spectrophotometry?
larger absorbance and lower light transmission to photocell
turbidity is measured with measurements of optical density (OD), what are the problems with this? 4
- has an infinite range of measurement
- only works if the cells are evenly distributed (no clumps)
- cuvette must not have scratches
- culture may need to be diluted when the cells are at very high density (so that actual doesn’t separate from theoretical)
why must we establish a standard curve to count turbidity value?
to compare the standard curve to another counting method
what is the other counting method that has to do with mass?
a specific volume of cells are concentrated, washed to remove media and concentrated and dried
then we weigh. takes a long time but is normally more reliable
