Miramar College

Biology 205 Microbiology

Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions
Background
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 and the dynamics of the microbial growth can be charted by means of a population growth curve, which is
constructed by plotting the increase in cell numbers versus time of incubation and can be used to delineate stages of
the growth cycle. It also facilitates measurement of cell numbers and the rate of growth of a particular organism under
standardized conditions as expressed by its generation time, the time required for a microbial population to double.
The stages of a typical growth curve (Figure 1) are:
1. Lag phase: When the cells are adjusting to their new environment. During this phase, cellular metabolism
is accelerated, resulting in rapid biosynthesis of cellular macromolecules, primarily enzymes, in 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 numbers. 2. Exponential phase: Under optimum 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 population, which doubles regularly until a maximum number of
cells is reached. The length of the log phase varies, depending on the organisms and the composition of
the medium, although the average may be estimated to last 6 to 12 hours. 3. Stationary phase: During this
stage, the number of cells undergoing division is equal to the number of cells that are dying. There is no
further increase in cell number and the population is maintained at its maximum level for a period of time.
The primary factors responsible for this phase are the depletion of some essential metabolites and the
accumulation of toxic acidic or alkaline end-products in the medium. 4. Death phase: Because of the
continuing depletion of nutrients and buildup of metabolic wastes, the microorganisms die at a rapid and
uniform rate. This decrease in population closely parallels its increase during the log phase. Theoretically,
the entire population should die during a time interval equal to that of the log phase. This does not occur,
however, since a small number of highly resistant organisms persist for an indeterminate length of time.

Figure 1: Growth curve for an hypothetical population. Note the four phases of growth: lag; exponential; stationary and
death.

Construction of a complete bacterial growth curve requires that aliquots of a 24-hour shake-flask culture be measured
for population size at intervals during the incubation period, however, such a procedure does not lend itself to a regular
laboratory session. This experiment is designed to include only the lag, log and possibly stationary phases of population
growth. Upon completion of this experiment, you will plot the data collected during this experiment by using two
values for the measurement of growth. The direct method requires that you use serial dilution to plate out cells at 30
minute intervals in order to calculate the number of colony forming units (CFUs) at a given time. The indirect method
uses spectrophotometric measurements of the developing turbidity at the same 30-minute intervals, as an index of
increasing cellular mass (assumed to correlate with an increase in the number of cells).
You will determine generation time with indirect methods by using data you collect, once it has been plotted onto a
graph like the one shown below. Indirect determination is made by simple extrapolation from the log phase as
illustrated in the figure below. Select two points on the optical density (OD) scale, such as 0.4 and 0.8, that represent a
doubling of turbidity. Using a ruler, extrapolate by drawing a line between each of the selected optical densities on the
ordinate and the plotted line of the growth curve. Then draw perpendicular lines from these end points on the plotted
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line of the growth curve to their respective time intervals on the abscissa (Figure 2). With this information, determine
the generation time using the formula GT =t(O.D. 1) - t(O.D. 2).

Figure 2: Graphical representation of the indirect method of measuring bacterial growth, optical density plotted vs. time.
Generation time is determined using two points within the exponential portion of the growth curve where the population
doubles in size, in this example: GT =t(O.D. 1) - t(O.D. 2) = 263 minutes - 222 minutes =43 minutes, so the generation time of this
hypothetical population is 43 minutes.

Introduction
In this experiment you will be determining the generation time of a bacterial culture. Because of the amount of
time needed for these cultures to reach the stationary phase of growth, the cultures will be started before class
begins. You will receive instruction as to when the cultures were started, what organism was used for the
inoculation, and what media these organisms were inoculated into. The class as a whole will be testing Escherichia
coli with each table having a culture started at different times. In this way, we hope to collect data that can be
plotted together and yield a growth curve sufficient to determine the generation time of this organism.
These cultures will be incubated throughout the experiment in a shaking incubator. The incubator will be set to
the optimal temperature for your specific bacterial species and will shake in order to keep your culture properly
aerated. These conditions will allow for optimal growth of your culture via aerobic respiration. You will take
control of this culture when lab begins.
At thirty minute intervals, you will take measurements of the cultures growth. You will do this directly using
serial dilutions and spread plating, and indirectly using OD readings. This means that you will be performing
the protocols outlined below at thirty minute intervals.
Objectives
1. Take metered spectrophotometric readings of a growing culture.
2. Perform serial dilutions and spread plating at metered intervals.
3. Plot growth data and determine generation time of a bacterial species.
Protocol
Team Supplies

Class Supplies

bacterial culture

shaking incubator

Spec20 spectrophotometer
sterile broth to zero the Spec20 spectrophotometer
ice bath
16 nutrient agar plates
4 test tubes containing 9.9 ml H2O
16 test tubes containing 9.0 ml H2O
200l mechanical pipetter and tips
1000l mechanical pipetter and tips
waste container for used tips
glass Petri dish and alcohol
spreader, plate spinner

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Day One: Setting Up

1. Properly zero your Spec 20, refer to Lab Exercise 10a: Using a Spectrophotometer and Pipetman if necessary.
2. LABEL YOUR MEDIA: For each time interval you will be taking measurements (e.g., time 0, time 120, time 150, etc.)
label five test tubes with the time period and 10-2, 10-3, 10-4, 10-5 and 10-6. Label four Petri dishes: 10-4, 10-5, 10-6 and 10-7.
You should have five test tubes and four Petri dishes for each time period.
Day One: Collection of Time Interval Specimens
Note: This section explains how to collect the specimens that will be diluted and plated in the following section.
3. AT 30 MINUTE INTERVALS (specified in step 3 above) perform the following steps. This procedure is also pictured
below:
1. Collect your flask from the shaking incubator.
2. Remove 100l of your bacterial culture from the flask and put it in the test tube labeled 10-2. Put this
test tube into your ice bath. Note: placing this tube on ice effectively halts binary fission and means
that you can take your time in setting up the remaining steps of the serial dilution and plating
experiment.
3. Give your flask a swish and then take an OD reading using the Spec 20. It is not necessary to remove
any of the culture for this step, simply tilt the culture to pour enough of the liquid to fill the attached
test tube with culture. Place this test tube into the Spec 20 and record the OD600nm.
4. Quickly put the culture back into the 37C shaking incubator.

Day One: Serial Dilution and Spread Plate Protocol

Note: These steps will be performed for EACH INCUBATION TIME, using the tubes collected during the Collection of
Time Interval Specimens section above.
1. Remove your 10-2 test tube from the ice bath, and mix the culture by rolling it carefully in your hands. Perform
the serial dilutions and plate a portion of each of these dilutions onto your four Petri dishes as directed in the
figure below.

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2.

Spread the bacterial inoculates onto each of the four plates using the protocol outlined in the figure below. Note:
heat is not the sterilizing step in this procedure- alcohol is. The only reason you are using heat is to remove the
alcohol once it has sterilized your spreader.
Note: There may be some residual liquid on the spreader even after you have flamed it. This is water, not alcohol.
Do not worry about removing it- its sterile. If you leave the spreader in the heat for longer than it takes to ignite the
alcohol, the spreader will be too hot to spread the culture without killing the cells.
Be sure also to keep a safe distance between the dish of alcohol and the flame so you do not cause the alcohol to
ignite in the dish. Never put a hot spreader into alcohol as the flash point of alcohol is relatively low (~56o F) and it
will easily ignite.

3.

After you have spread the inoculates onto the Petri dishes, leave them agar side down in the 37C incubator.

Day Two: CFU counts

1. Collect your plates from the 37C incubator. Count the number of distinct colonies on each plate.
2. For each time set, keep only those plates which contain between 30-300 colonies, discard all the rest. If more than
one plate contains 30-300 CFUs, average the numbers and record only one for each time set.
3. Determine the number of CFUs/ml of the original culture for each of the time sets.
Results and Data Analysis
1. Record the optical densities and corresponding cell counts in the chart below.
Clock Time

Incubation Time
(min. past)a

Optical Density
(600nm)

CFU Counts
(CFU/ml)

This column records the actual number of minutes passed from the time zero inoculation.

The following questions can be completed after Lab Exercise 14: Data Analysis and Presentation I.
2. Plot the CFUs/ml on the Y axis and incubation times on the X axis of the provided semi-log paper (you may also do
this using computer software). Plot the OD600nm on the Y axis and incubation times on the X axis of the provided
graph paper.
3. Identify the log phase for each of the graphs and determine the generation time of your bacterial culture using
both the indirect and direct methods described in the introduction to this lab exercise. Remember that the log
phase is represented by the straight-line portion of the curve.
Discussion
1. Does the term growth convey the same meaning when applied to bacteria and to multicellular organisms? Explain.
2. Why do variations in generation time exist:
a. among different species of microorganisms?
b. within a single microbial species?
3. Is generation time a useful parameter to indicate the types of media best suited to support the growth of a specific
organism? Explain.

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