3.2: Measuring Microbial Growth Flashcards
(19 cards)
How do microscopic counts using a petroff-hausser counting chamber work?
- cells can be counted by direct microscope observations using a petroff-hausser counting chamber.
- each square corresponds to a calibrated volume.
-usually 0.2 mm x 0.2 mm x 0.02 mm deep, so =0.0008 mm3 (or 0.0008 ul).
What’s the formula to calculate microscopic count using petroff-hausser counting chamber?
- divide # of cells/ by the volume (0.0008 ul) x 1000ul (per ml) = cells/ml
what are the 2 problems associated with direct microscopic counts?
- Precision is difficult to achieve:
-small sample volumes and random distribution of cells make microscopic counts error prone
-many counts are required - Counts both live and dead cells -total count
-we only want to know live count not total count
what’s the solution of direct microscopic counts including total count (dead and alive cells)?
- stain the live cell with DAPI
- only live cells containing DNA will fluoresce and be counted.
how does flow cytometry method for counting cells work?
- liquid is passed through a very narrow passage
- only one cell can pass through at a time
- a laser is used to detect light scattering
- each scattering event =1 cell
what type of samples are flow cytometers used for counting cells
this is a method for counting cells in liquid samples
what’s a downside of using flow cytometry to count cells
the laser detects light scattering and cannot tell if it scans alive or dead cells, counts total cells.
what is the most common way for counting cells
- viable cell counts (plate counts)
how do viable cell counts work?
- measures only living cells that are able to grow and form a colony
- sample is normally diluted prior to plating
- assumes that each colony that forms came from one viable cell in the original sample (one cfu).
what does the method of viable cell counts assume
- assumes each colony that forms came from one viable cell in the original sample (one cfu).
what are the problems associated with viable counts?
- Labour and material intensive: Requires serial dilutions, plating, and incubation to count a single sample
- plate counts can be highly unreliable when used to count bacteria present in natural samples (like soil and water, because they may not be able to grow on T-soy)
- cells that are stuck together will be counted as a single colony forming unit
- only those organisms that can form colonies on the selected media will grow, so you can only count the types of bacteria that can grow on the selected medium!
what is the great plate count anomaly?
- direct microscopic counts of natural samples reveal far more organisms than those on plates.
- this is bc, as modern genomic techniques suggest, only 1-10% of microbial diversity is culturable
why do direct microscopic counts of natural samples reveal far more organisms than plate counts?
- microscopic methods count dead cells, whereas viable methods do not
- different organisms may have vastly different requirements for growth
- we do not know the specific requirements for all organisms (especially in natural samples, may not know exactly whats in there)
how does spectrometry measure microbial growth?
- uses a spectrophotometer to measure turbidity (how cloudy it is) of a sample
- measurement is referred to as optical density (OD)
- it’s a indirect measurement of cell density
-based on the fact that bacteria in suspension will scatter and block the passage of light
more cells = more cloudy =higher OD
what are the problems with optical density measurements?
- does not distinguish live and dead cells
- relatively insensitive - need at least about 10^8 cells/ml to measure as OD
- has a finite linear range (once it reaches a certain point you can’t measure anything, too cloudy)
- only works if the cells are evenly distributed throughout the medium (no clumps or biofilms)
- bc it’s an indirect count it doesn’t actually tell you how many cells are in your suspension, just how amount changes.
what must be made first to relate direct count to optical density?
a standard curve must first be established graphing another counting method (x-axis) vs OD (y-axis).
how can a standard curve be used?
OD can be used to estimate cell number
what’s the limitation of a standard curve
It is only good for one particular organism in one particular set of growth conditions, (cannot be used again for different condition).
how is cell dry weight used to measure cell growth
- known volume of culture centrifuged to concentrate the cells (you’re left with pellets)
- it’s washed to remove media components
- centrifuged again and dried
- the dried cells are weighed and the weight is expressed as mg cells (dry weight)/ml of culture.
