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 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