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Measures of Microbial Populations. Quantifying the unseen. Variety of Approaches. Direct Cell Count (DCC). Petroff-Hauser Counting chamber (Hemocytometer) and Phase Contrast Microscope Sensitvity down to 10 5 cells per ml Live cells and dead cells counted equally Motility?.

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measures of microbial populations

Measures of Microbial Populations

Quantifying the unseen

direct cell count dcc
Direct Cell Count (DCC)
  • Petroff-Hauser Counting chamber (Hemocytometer) and Phase Contrast Microscope
  • Sensitvity down to 105 cells per ml
  • Live cells and dead cells counted equally
  • Motility?
viable cell count
Viable Cell Count
  • Usually requires serial dilution of culture (sample)
  • Followed by plating technique
  • Sensitivity down to 300 cells per ml (or less)
  • Counts only “live” cell – Viable
  • Assumes each colony forms from a single cell
dilution and dilution factors
Dilution and Dilution Factors
  • A small amount of liquid is dispersed evenly into a volume of diluent (e.g. PBS)
  • The new population density relative to the original is expressed as the dilution factor (DF)
  • DF is always less than 1, and a unit-less number (ratio)
serial dilutions
Serial Dilutions
  • To greatly reduce a population density, dilutions are made from dilutions!
  • This series of dilutions is referred to as a serial dilution
  • The serial dilution factor is just the product of the individual dilution factors
plating
Plating
  • Samples and their dilutions are applied to some solid media so that individual colonies will arise
  • Spread plate technique and pour plate technique
counting spread plates
Counting Spread Plates
  • Quebec Colony Counter
  • Each colony may arise from a single cell or a group of cells (chain, tetrad, etc.)
  • Colony Forming Unit – CFU
  • Accurate range 30-300 CFU
    • <30 sample size too small
    • >300 too close to individuate TNTC
optical density a bright idea
Optical Density – A Bright Idea
  • Spectrophotometer is used to measure how much light is blocked out by bacteria in suspension
  • We will use OD units (absorbance) rather than Klett Units
correlation between od and vcc
Correlation Between OD and VCC
  • Due to size and shape the amount of light blocked out is unique to each species
  • One must develop a correlation between Absorbance and CFU per ml (basically the slope of graph b)
today s exercise
Today’s Exercise
  • Perform dilution and plating as per lab handout
  • Measure theOD600 of your culture
  • Divide CFU per ml (result 1) by the OD600 (result 2) to obtain CFU per ml per OD
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