Bacterial Growth curve
Aim
To
plot the growth curve of an aerated bacterial culture and determine the
generation time
Principle
Bacterial
population growth studies require inoculation of viable cells into a sterile
broth medium and incubation of the culture under optimum temperature, pH and
gaseous conditions. Under these conditions, the cells will reproduce rapidly.
The increase in the cell number versus the time of incubation is plotted to
obtain the growth curve.
The curve can be used to explain the
stages of the growth cycle. It also helps in the measurement of cell numbers
and the rate of growth of a particular organism under standard conditions. The
growth rate is expressed by its generation time, the time required for the
microbial population to double.
The stages of a typical growth curve
are:
1. LAG
PHASE
During this stage, cells are adjusting to
their new environment. Cellular metabolism is accelerated resulting in rapid
biosynthesis or preparation for the next phase of the cycle. Although the cells
are increasing in size, there is no cell division and therefore no increase in
number.
2. LOGARITHMIC
PHASE
Under optimal nutritional and physical
conditions, the physiologically robust cells reproduce at a uniform and rapid
rate by binary fission. Thus there is a rapid exponential increase in the
population which doubles regularly until a maximum number of cells is reached.
The time required for the population to double is the generation time. The
length of the log phase varies depending on the organisms and the composition
of the medium. The average may be estimated to last for 6-12 hours.
3. STATIONERY
PHASE
During this stage some cells are dying.
Therefore, there is no further increase in cell numbers and the population is
maintained at its maximum level for a period of time. The primary factors
responsible for this stage are the depletion of some essential; metabolites and
accumulation of toxic, acidic or alkaline end products in the medium.
4. DECLINE
/DEATH PHASE
Because of the continuing depletion of
nutrients and buildup of metabolic wastes, the microorganism die at a rapid and
uniform rate. The decrease in population closely parallel its increase during
log phase. Theoretically, the entire population should die during a time
interval equal to that of the log phase. This does not occur because a small
number of highly resistant organisms persist for an indeterminate length of time.
Construction
of a completed bacterial growth curve requires that aliquots of a 24-hour
shake-flask culture be measured for population size at intervals during the
incubation period. Such a procedure does not tend itself to a regular
laboratory session. Therefore, this experiment follows a modified procedure
designed to demonstrate only log and lag phases, the curve will be plotted by
using the two values for the measurement of growth. The direct method requires
enumeration of viable cells in serially diluted samples of the test culture
taken at 30 minutes interval.
The indirect method uses
spectrophotometric measurement of the developed turbidity at the same 30
minutes as an index of increasing cellular mass. Indirect determination of
generation time is made by simple extrapolation from the log phase as
illustrated in the figure. Select two points on the optical density scale such
as 0.2 and 0.4 that represent doubling of the turbidity. Using a regular scale
extrapolate, draw a line between each of the selected optical density on the
coordinates of the plotted line of the growth curve. Then draw perpendicular
lines from these end points on the Y axis to their respective time intervals on
the X axis. Generation time is calculated as,
GT = t OD0.4- t OD0.2
Materials Required
1. Culture-
10-12 hours (log phase) nutrient broth culture of Escherichia coli
2. Media-
100 ml nutrient broth in 250 ml flask
3. Equipments-
37oC water bath shaker, incubator, spectrophotometer, Bunsen burner,
glassware marking pencil
Procedure
1. Nutrient
broth was prepared and 100 ml medium dispersed into series of 250 ml conical
flask and sterilized at 121oC and 15 lbs pressure for 15 minutes.
2. Then,
the flask was aseptically inoculated with 5 ml of overnight E. coli culture and kept in a shaker for
constant aeration and agitation.
3. The
flasks were removed at an interval of I hour and samples transferred
aseptically to a clean cuvette using sterile pipette.
4. Optical
density was read using a spectrophotometer at 600 nm and readings were
tabulated.
5. The
readings were then plotted on on a graph sheet with Optical density values
representing growth on the Y-axis and incubation time on the X-axis.
Observation and Result
The optical density values for each
hour time interval was tabulated. Growth curve was plotted with OD at 600 nm on Y-axis versus incubation time on X-axis. Generation time of E. coli was calculated by extrapolation
from the plotted curve.
GT = t OD0.4- t OD0.2
GT = 180 minutes – 150 minutes = 30
minutes
Observation (on left-hand side)
|
Time
(Hours) |
Optical
Density at 600 nm |
|
0 |
... |
|
0.5 |
... |
|
1 |
... |
|
2 |
... |
|
3 |
... |
|
4 |
... |
|
5 |
... |
|
6 |
... |
|
7 |
... |
|
8 |
... |
|
24 |
... |
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