Lecture Set 3 : Part 2 Flashcards
what are microscopic counts?
-cells can be counted by direct microscopic observations
-using a Petroff-Hausser counting chamber (can be other names)
how does a counting chamber work?
-a slide with large squares on it that correspond to a calibrated volume
-it gives an estimate of the total population
-cells must be equally spread across the slide
-typically multiple large squares are counted and averaged (accounts for clumping/variability)
what are some problems with microscopic counts?
-precision can be difficult to achieve (small sample volumes + random distribution = error prone)
-counts both live and dead cells because you cannot differentiate so it gives a total count
what is a solution that allows for only live cells to be counted within microscopic counts?
-you can stain samples with DAPI
-only live cells containing DNA will fluoresce (DAPI binds to DNA)
what is flow cytometry?
-method for enumerating cells in liquid samples
-liquid is passed through a very narrow passage where only 1 cell can pass at a time to be counted
-a laser detects light scattering (each scattering event = 1 cell)
-expensive option
-cells can be sorted into populations
-still can count both live and dead cells (dead cells can still scatter light)
what is a viable cell count?
-plate counts
-measure only living cells that are able to grow and form a colony
-sample gets diluted typically before plating
-spread plate or pour plate
-must make the assumption that each colony that forms came from one viable cell in the original sample (1 cfu)
what are the problems with viable cell counts?
-it is labour and material intensive
-requires serial dilutions + plating + incubation to enumerate a single sample
-spot plating could be less, but also is less accurate
-only organisms that can form colonies on the selected media will grow and can be counted
-plate counts can be highly unreliable when used to count bacteria present in natural samples (difficult to get them to be homogenous, ex: soil + water) (also may not grow on media, only 1-10% of microbial diversity is culturable)
what is better method to count natural samples?
-direct microscopic counts
-reveals more organisms than plates can as we do not know the specific requirements for growth of all organisms (different organisms may have vastly different growth requirements)
if we wanted to count only dead cells, what counting method could be used?
-microscopic counts
what is the method of counting using spectrophotometry?
-measures the turbidity (optical density) of a sample
-measured at a specific wavelength
-indirect measurement of cell density as it is based on the idea that bacteria will scatter light
-gives an immediate reading
what are the problems with optical density measurements?
-does not distinguish between live and dead cells
-needs to meet a minimum threshold of at least 10^8 cells/ml to give a reading (any less gives 0)
-has a finite linear range (portion where this relationship exists, between 0.1-1.0) (can be solved with diluting)
-cells must be evenly distributed (cannot form clumps or biofilms, skews readings)
-is an indirect count (does not tell you how many cells are in your suspension) (need a standard curve to get a count) (if cells are larger they can scatter more light)
what is the standard curve?
-used to relate a direct cell count to optical density
-established using another counting method (viable cell counts or microscopic counts)
-once generated, OD can be used to estimate a cell number
-only good for one particular organism in one particular set of growth conditions
what is another method used to measure cell growth?
-cell dry weight
how do you get the cell dry weight?
-a known volume of culture is centrifuged (spun) to concentrate the cells
-gets washed to remove media components and then centrifuged again
-dried cells are then weighed and expressed as mg cells/ml of culture
what can you also measure similarly to a cells dry weight?
-the mass of specific cell constituents (protein, DNA, etc)
-must assume that cell constituents increase proportionally with an increase in cell number
-expressed as mg of ___/ml of culture
