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Measures of Microbial Populations

Explore various techniques like Direct Cell Count, Viable Cell Count, Dilution Factors, Plating, Optical Density, and more for measuring microbial populations accurately. Learn about dilution and plating procedures, factors affecting colony formation, and the correlation between optical density and viable cell count. Engage in today's lab exercise to practice these methods effectively.

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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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