Chapter4CellCount

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Lecture 9 - Growth
Measuring Growth
Learning Objectives
Be able to sketch and label a bacterial growth curve (x- and y-axis as well as phases).
Explain what happens in each phase of a bacterial growth curve.
Be able to use the growth equation to model bacterial growth.
Explain the difference between primary and secondary metabolites, and give an example of each.
Explain why it takes a very long time for 100% of the bacteria in a culture to die.
Cite three differences between a continuous culture and a batch culture.
Know HOW and WHEN to quantitatively measure bacterial growth by each of the following methods
Direct counts
Petroff-Hauser chamber
Coulter counter
Viable (Indirect) counts
Dilution series + pour plate
Dilution series + spread plate
Membrane filtration method
Most probable number method
Biomass measurements
Turbidity
Total cell mass
Know how bacterial growth can be quantified by measuring the accumulation of primary metabolites
such as acid and gas, or by measuring ATP formation.
Vocabulary
lag phase
exponential phase
stationary phase
death phase
growth rate constant
generation time
primary metabolite
secondary metabolite
persister cells
batch culture
continuous culture
chemostat
direct cell count
Petroff-Hauser chamber
Coulter counter
viable cell count
pour plate
spread plate
membrane filter
most probable number (MPN)
turbidity (= optical density)
biochip
conductivity
Durham tube
luciferase
The Bacterial Growth Curve
Growth equation based on binary fission
(same as the compound interest formula)
Nt = N0emt
N = number of cells
m = growth rate constant ( / hr.)
t = time (hr)
How long will it take one cell dividing at a rate
of 0.5 per hour to become a colony of
100,000 cells?
100,000 = (1) e0.5t
t = 23 hrs
What is the growth rate if it takes a cell 45
minutes to divide?
Nt = N0emt
2 = (1) em(45/60)
ln2 = 0.75m
m = 0.92 / hr
Production of metabolic products
follows growth curve kinetics
Decline phase can be VERY long,
especially in mixed cultures
especially due to persister cell survival
Batch versus Continuous Culture
• Batch is in a flask – growth stops when
– wastes build up
– nutrients run out
– the cells become too crowded to get O2
• Continuous is in a chemostat
– nutrients continually added at slow rates
– bacteria perpetually in exponential phase
– growth rate determined by nutrient resupply rate
Counting Bacteria
• Direct cell counts (all cells, live and dead ones)
– Petroff-Hauser chamber
– Coulter counter
• Viable cell counts (only live cells)
– Dilution and plating
• pour plate
• spread plate
– filter plating
– most probable number method
• Biomass measurements
– turbidity
– weigh cells
• Measure cell products
– acid
– gas
– ATP
Petroff-Hauser chamber allows
microscopic counts of all cells
within a grid square of known size
Coulter counter measures
interruption of electric current as
cells pass through counter
Dilution and plating schemes count
all cells that can form colonies
Spread plates put diluted culture
ON TOP OF the agar
Pour plates put diluted culture
WITHIN the agar
Membrane filtration method allows
large volumes (100s of ml) to be
counted
MPN is a statistical method based on +/- growth
in a large number of tubes at 3 different dilutions
Turbidity is based on OD
measurements with a
spectrophotometer
• more cells = more light scattered = higher OD
• barely any scatter ~107 cells/ml
• need to correlate OD with cell number for a
given species
Weigh cells directly
• rules of thumb:
– “typical” cell weighs ~1 pg wet
– cell is ~70% water
– cell is ~50% protein
– You can harvest ~ 1g of cells (wet) from a liter
of culture in a rich, complex medium
Measurement of cell products
depends on metabolism of cells
being studied
• acid from fermentation – easy to measure
with pH electrode
– micro-scale applications in food science
Gases measured in Durham tube
or by pH if gas is acidic
ATP can be measured by
luciferase reaction
• sometimes done to see if any viable cells
remain in a culture (no ATP = no
metabolism = dead)
• not quantitative since cells produce
different amounts of ATP in different
phases of growth
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