Transcript Measures of Microbial Populations

Measures of Microbial
Populations
Quantifying the unseen
Variety of Approaches
Direct Cell Count (DCC)
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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
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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
DF 
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VolumeTransferre d
VolumeTotal

VolumeTransferre d
VolumeTransferre d  VolumeDiluent
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
DFSerial  DF1  DF2
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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
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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
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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
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<30 sample size too small
>300 too close to individuate
TNTC
VCC
CFU
VCC 
VolPlated  DF
159CFU
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(1m l)(103 )
5 CFU
 1.59  10
ml
Optical Density – A Bright Idea
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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
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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.
2.
3.
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