Download Lab 18 – growth curve

Measurement of Bacterial
Growth
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Growth is an orderly increase in the quantity of
cellular constituents. It depends upon the ability
of the cell to form new protoplasm from nutrients
available in the environment. In most bacteria,
growth involves increase in cell mass and number
of ribosome, duplication of the bacterial
chromosome, synthesis of new cell wall and
plasma membrane, partitioning of the two
chromosomes, septum formation, and cell
division. This asexual process of reproduction is
called binary fission.
Measurement of Bacterial Growth
Growth in Batch Culture
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“Growth” is generally used to refer to the
acquisition of biomass leading to cell division, or
reproduction
A “batch culture” is a closed system in broth
medium in which no additional nutrient is added
after inoculation of the broth.
Generation time
Time taken for a cell population to double in
numbers and thus equivalent to the average length
of the cell cycle
Growth in Batch Culture
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Typically, a batch culture passes through four
distinct stages:
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–
–
Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage
Growth in Batch Culture
Table 2. Generation times for some common bacteria under
optimal conditions of growth.
Medium
Generation Time
(minutes)
Glucose-salts
17
Bacillus megat
Sucrose-salts
erium
25
Streptococcus
lactis
Milk
26
Streptococcus
lactis
Lactose broth
48
Bacterium
Escherichia
coli
Staphylococcus Heart infusion
aureus
broth
27-30
Table 2. Generation times for some common bacteria under
optimal conditions of growth.
Lactobacillus
acidophilus
Milk
66-87
Rhizobium
japonicum
Mannitol-saltsyeast extract
344-461
Mycobacteriu
m
Synthetic
tuberculosis
792-932
Treponema
pallidum
1980
Rabbit testes
The Bacterial Growth Curve
Growth in Continuous Culture
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A “continuous culture” is an open system in which
fresh media is continuously added to the culture at a
constant rate, and old broth is removed at the same rate.
This method is accomplished in a device called a
chemostat.
Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as the
nutrient feed is maintained.
Factors that Influence Growth
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Temperature
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–
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Most bacteria grow throughout a range of approximately
20 Celsius degrees, with the maximum growth rate at a
certain “optimum temperature”
Psychrophiles: Grows well at 0ºC; optimally between
0ºC – 15ºC
Psychrotrophs: Can grow at 0 – 10ºC; optimum between
20 – 30ºC and maximum around 35ºC
Mesophiles: Optimum around 20 – 45ºC
Moderate thermophiles: Optimum around 55 – 65 ºC
Extreme thermophiles (Hyperthermophiles):
Optimum around 80 – 113 ºC
Factors that Influence Growth
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pH
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Acidophiles:
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Neutrophiles
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Grow optimally between ~pH 0 and 5.5
Growoptimally between pH 5.5 and 8
Alkalophiles
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Grow optimally between pH 8 – 11.5
Factors that Influence Growth
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Salt concentration
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Halophiles require elevated salt concentrations to
grow; often require 0.2 M ionic strength or greater
and may some may grow at 1 M or greater; example,
Halobacterium
Osmotolerant (halotolerant) organisms grow over a
wide range of salt concentrations or ionic strengths;
for example, Staphylococcus aureus
Factors that Influence Growth
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Oxygen concentration
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Strict aerobes: Require oxygen for growth (~20%)
Strict anaerobes: Grow in the absence of oxygen; cannot
grow in the presence of oxygen
Facultative anaerobes: Grow best in the presence of
oxygen, but are able to grow (at reduced rates) in the
absence of oxygen
Aerotolerant anaerobes: Can grow equally well in the
presence or absence of oxygen
Microaerophiles: Require reduced concentrations of
oxygen (~2 – 10%) for growth
Table 1. Some Methods used to measure bacterial growth
Method
Application
Comments
Direct microscopic
count
Enumeration of
Cannot distinguish living
bacteria in milk or
from nonliving cells
cellular vaccines
Viable cell count
(colony counts)
Enumeration of
bacteria in milk,
foods, soil, water,
laboratory
cultures, etc.
Very sensitive if plating
conditions are optimal
Turbidity
measurement
Estimations of large
numbers of
bacteria in clear
liquid media and
broths
Fast and nondestructive, but
cannot detect cell
densities less than 107
cells per ml
Table 1. Some Methods used to measure bacterial growth
Measurement of total
cell yield from
very dense
cultures
only practical application
is in the research
laboratory
Measurement of
Biochemical
activity e.g. O2
uptake CO2
production, ATP
production, etc.
Microbiological
assays
Requires a fixed standard
to relate chemical
activity to cell mass
and/or cell numbers
Measurement of dry
weight or wet
weight of cells or
volume of cells
after centrifugation
Measurement of total
cell yield in
cultures
probably more sensitive
than total N or total
protein measurements
Measurement of total
N or protein
Calculation of Generation Time
Because of the very large differences in the number of cells
present at the peak and at the start/end of the experiment, it's hard
to see what's going on from this graph.
It's much easier to see the whole experiment if you plot the
number of viable cells on a logarithmic scale (or more simply,
plot the log of cell number).
Calculation of Generation Time
the log plot
Calculation of Generation Time
As you can see, the indirect method of counting (optical density)
closely parallels the direct method (viable count). (At later time
points, you can see that the number of viable cells declines faster
than the optical density of the culture.
It will be even easier to see the results if we concentrate on the
first 12 hours of the experiment.
Calculation of Generation Time
The graph of the results reveals FOUR distinct phases which
occur during the growth of a bacterial culture.
Calculation of Generation Time
When growing exponentially by binary fission, the increase in a
bacterial population is by geometric progression. If we start with
one cell, when it divides, there are 2 cells in the first generation, 4
cells in the second generation, 8 cells in the third generation, and
so on. The generation time is the time interval required for the
cells (or population) to divide.
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
G=
t
3.3 log b/B
Calculation of Generation Time
Example: What is the generation time of a bacterial population
that increases from 10,000 cells to 10,000,000 cells in four hours of
growth?
G=
t_____
3.3 log b/B
G = 240 minutes
3.3 log 107/104
G = 240 minutes
3.3 x 3
G = 24 minutes
Mean Generation Time
and Growth Rate
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The mean generation time (doubling time) is the
amount of time required for the concentration of
cells to double during the log stage. It is
expressed in units of minutes.
1
Growth rate (min-1) = mean generation time
Mean generation time can be determined directly
from a semilog plot of bacterial concentration vs
time after inoculation
Mean Generation Time
and Growth Rate