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Measures of Microbial Populations

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

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  1. Measures of Microbial Populations Quantifying the unseen

  2. Variety of Approaches

  3. 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?

  4. 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

  5. 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)

  6. 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

  7. Plating • Samples and their dilutions are applied to some solid media so that individual colonies will arise • Spread plate technique and pour plate technique

  8. 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

  9. VCC

  10. 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

  11. 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)

  12. 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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