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 105 cells per ml Live cells and dead cells counted equally Motility?

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

DiluentdTransferre

dTransferre

Total

dTransferre

VolumeVolume

Volume

Volume

VolumeDF

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

21 DFDFDFSerial

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 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

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

VCC

ml

CFU

ml

CFU

DFVol

CFUVCC

Plated

5

3

1059.1

)10)(1(

159

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

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

1. Perform dilution and plating as per lab handout

2. Measure theOD600 of your culture

3. Divide CFU per ml (result 1) by the OD600 (result 2) to obtain CFU per ml per OD