Measures of microbial populations
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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

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Measures of microbial populations

Measures of Microbial Populations

Quantifying the unseen


Variety of approaches

Variety of Approaches


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


Measures of microbial populations

VCC


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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