Lesson plan

Bacterial Growth Curves:

Analysis through OD₆₀₀ measurements
Introduction Bacterial growth is characterized through four different phases:
Bacteria are single cell organisms often characterized by lag, log/exponential, stationary, and death phases (Figure 1).
their size, shape, vulnerability to specific antibiotics, and The lag phase is the first phase of bacterial growth and outlines
pathogenicity, among other properties.¹ Under the appropriate the time it takes for bacteria to begin multiplying. In this phase,
conditions (e.g., temperature, nutrients), bacteria can reproduce the bacteria begin to form the enzymes required to initiate
through binary fission, producing more bacteria over time.¹,² growth. As described previously, the bacterial growth arises
Microbiologists will often study bacterial growth to better from binary fission, where one bacterium divides into two
understand the different growth phases for a specific strain, bacteria, which in turn produces two more bacteria each, and
such as the time it takes to double the population; this so on. This behavior will result in an exponential growth in the
“doubling time” is a characteristic specific to the species and number of bacteria formed as a function of time and outlines
strain analyzed.¹ Additionally, analysis of bacterial growth can the second growth phase: exponential or log phase.² During
aid in determining the best experimental conditions for growing this phase, nutrients within the growth medium (e.g., broth,
bacterial colonies (aggregates of clustered bacteria) according agar) are being consumed. At some point, enough nutrients
to the needs of the scientist. have been consumed such that the bacteria can no longer
keep reproducing at an exponential rate, reaching a point of
diminishing returns where the number of living bacteria no
Stationary longer changes (stationary phase). Once the nutrients are
Phase diminished, and in the presence of waste byproducts produced
Log (# of Bacterial Cells)

during growth, the bacteria cannot survive indefinitely and will

Death
Phase begin to die, leading to the death phase.
Log or
Exponential Phase There are several different methods that can be employed to
monitor the growth of bacterial colonies, including detecting
Lag nucleic acid sequences specific to the bacteria being
Phase
grown and detecting of the structure of the bacteria through
Time antibodies, among others.¹ One commonly employed method
involves growing cultures on solid growth media from aliquots
Figure 1. Example diagram for a typical bacterial growth curve.
of the original growth culture collected at different time
intervals during the experiment. The number of viable (living)
colonies are then counted and reported for each time point.
This method allows for the direct monitoring of the number
of bacteria grown at a given time and is specific to living
bacteria as dead colonies are unable to grow. Unfortunately,
this technique is time-consuming as the bacteria must grow
overnight before it can be counted.
Another frequently used technique to monitor bacterial
Experimental
growth involves UV-Visible spectroscopy. In principle, this
technique uses light across the UV-Visible range of the
Materials
electromagnetic spectrum to probe electronic transitions
• Thermo Scientific™ GENESYS™ 40 or 50
within a given analyte (molecule or biomolecule). Typically,
UV-Visible Spectrophotometer
data is reported as an absorption spectrum which
describes these transitions. However, in the context of • Disposable cuvettes (plastic cuvettes are appropriate)
bacteria this technique is different. As the bacterial colonies • Lint-free lab wipes
grow, they become large enough to scatter light, which
• Escherichia coli (E. coli), Strain: K-12
appears as an absorption feature across the entirety of the
UV-Visible spectrum. With an increase in the number of • Luria Bertani (LB) Broth (1X)
colonies, the measured “absorbance” will increase as well. • 50 mg/mL streptomycin
By measuring the “absorbance” at long wavelengths
• 10% Bleach solution
(600 nm is typically used by convention – OD₆₀₀), the growth
of the bacteria can be monitored much more quickly than • Metal or disposable inoculating loops (10 μL)
using the viable colony counting method, giving an almost
• Metal spreader
instantaneous observation of growth.
• LB Agar plates (with 50 μg/mL streptomycin)
It is important to keep in mind that this is not a true
• 250 mL baffled flask
measurement of the absorbance of a sample, but an
indirect measurement of the scattering of light off the newly • 2 L baffled flask
formed bacterial colonies. Additionally, this method will • Incubator
measure scatter off any material capable of scattering light
• Shaker
indiscriminately, including both living and dead bacteria.
While this method can provide a faster method of monitoring • Bunsen burner or alcohol burner (optional)
bacterial growth, it will inherently have error in the analysis
• Igniter (e.g. matches, lighter, spark igniter)
compared with counting viable colonies.
• Pipettor with appropriate pipette tips
In this experiment, you will be monitoring the growth of
• Permanent marker (preferably with a fine tip)
Escherichia coli (E. coli) K12 cultures as a function of time
through two different methods: viable colony counting
Safety
from cultures grown on plates and OD₆₀₀ measurements.
Note, in this lab students will be using a source of heat to
Using the data from both methods, you will construct a
sterilize inoculating loops. Care should be taken to ensure
growth curve for the formation of E. coli as a function of time.
students and instructors do not burn themselves or allow the
The experiment described herein is loosely based on the
flame to become uncontrolled. Instruct students on proper
experiment outlined by McKernan.³
fire safety, as well as applicable fire extinguishing methods.
Additionally, students should be wearing proper personal
References
1. McGoverin, C.; Steed, C.; Esan, A.; Robertson, J.; Swift, S.; Vanholsbeeck, F., Optical protective equipment, including gloves, when performing this
Methods for Bacterial Detection and Characterization, APL Photonics, 2021, 6. experiment or disposing of materials.
2. Harvey, R. A.; Champe, P. C.; Fisher, B. D., Microbiology Second Edition; Lippincott
Williams & Wilkins, 2006.
3. McKernan, L. N., Using a Simple Escherichia coli Growth Curve Model to Teach the
Scientific Method, Am. Biol. Teach., 2015, 77, 357-362.
Instructions A5. Repeat step A4 on a second agar plate.
Part A – Plating Bacterial Colonies A6. Using just a sterile inoculating loop (no E. coli), spread
A1. Clean/disinfect the lab bench. on the final agar plate without E. coli, repeating the
procedure outlined in step A4. This will serve as your
A2. Obtain (3) agar plates and the E. coli strain. Label the
negative control.
bottom of the plates appropriately (e.g. student initials,
date, E. coli strain) A7. Extinguish the flame.

NOTE: When removing the lid from the agar plate, A8. Place covers on each agar plate and place the plates in
ensure the lid is placed face up on the lab bench the incubator. The plates must be placed in the incubator
so as to avoid introducing any additional bacteria upside down.
into the agar plate.
A9. Set the temperature to 37 ˚C and allow to
incubate overnight.
A3. Set up a Bunsen burner or alcohol lamp.

a. Ensure safe lab procedures are followed when

handling an open flame. Part B – Overnight Culture
NOTE: Skip this step if using disposable B1. Following incubation, check for colony growth on
inoculating loops. your agar plate. Check with your instructor to make
sure the colonies are acceptable to be used for the
A4. Using a sterile inoculating loop, pick and spread the E. coli overnight culture.
on an agar plate. See Figure 2a for a visual description for
B2. Clean/disinfect the lab bench.
spreading bacteria colonies on a plate for growth.
B3. Fill a 250 mL baffled flask with 50 mL of LB broth.
Instructions for Spreading/Streaking Plates:
B4. Calculate the volume of 50 mg/mL streptomycin
a. Using the sterile inoculating loop, pick the E. coli needed to result in a final concentration of 50 μg/mL
colony and spread it in one quadrant of the agar plate. in 50 mL of solution:
Running the inoculating loop over the flame will
sterilize the loop (see Figure 2b).

b. Turn the plate 90˚, obtain a new disposable loop or

re-sterilize the metal inoculating loop and wait for the
loop to cool.

c. Using the cooled, sterilized loop, spread from the first

quadrant into the next quadrant.

d. Turn the plate another 90˚, obtain a new disposable B5. Add the calculated amount of 50 mg/mL streptomycin to
loop or re-sterilize the metal inoculating loop and wait the LB broth.
for the loop to cool.
B6. From one of the plates, pick a single bacterial colony using
e. Using the cooled, sterilized loop, spread from the a sterile inoculating loop.
second quadrant into the next quadrant.
B7. Dip the inoculating loop with the picked colony in the
f. Turn the plate another 90˚, obtain a new disposable baffled flask and stir it a little.
loop or re-sterilize the metal inoculating loop and wait
B8. Remove the loop and properly dispose of it or, if it is
for the loop to cool.
not disposable, flame the inoculating loop as shown in
g. Using the cooled, sterilized loop, spread from the third Figure 2b to sterilize. Allow to cool before putting away.
quadrant into the next quadrant.
B9. Place the flask in the incubator and allow to incubate at
NOTE: If using disposable inoculating loops, sterilization 37 ˚C overnight (maximum of 16 hours). Ensure the solution
is not necessary. Dispose of the loop after each use, is able to shake during the incubation. The shaker should
then use a new one each time the plate is turned. be set to 300 rpm.

a b
Incubate at
Sterilize Sterilize Sterilize 37° C
loop loop loop overnight

Rotate Rotate Rotate

plate 90° plate 90° plate 90°

Figure 2 - (a) Scheme demonstrating streaking a plate. (b) Depiction of inoculating loop sterilization. Images created with BioRender.com.
Part C – Bacterial Growth Curve C10. Using 2.0 mL of the reserved batch culture, fill a 1.0 cm
C1. Clean/disinfect the lab bench. cuvette and place in the UV-Visible spectrophotometer
sample holder.
C2. Turn on the UV-Visible spectrophotometer.
C11. Press “Measure” to measure the absorbance of the
C3. Open the “Fixed” Application and select the following sample. Record the measured absorbance and growth
instrument parameters: time in Table 1 in the Lab report section of this experiment.

a. Wavelength (λ₁): 600 nm C12. Repeat steps C8 – C11 every 20 min. until the end of the
lab period.
b. Factor (F₁): 1.000
C13. Add bleach to the batch and overnight cultures and
c. ABS mode properly dispose of the solution.

C4. Calculate the volume of 50 mg/mL streptomycin C14. Using the aliquots collected at 0, 60, 120, 160 min, serially
needed to result in a final concentration of 50 μg/mL in dilute 1:10 with LB broth for a total of 6 serial dilutions per
612 mL of solution: time point as described below:

a. Dilution 1: Add 1.0 mL of batch culture to 9.0 mL

of LB broth. Cap and mix by shaking.

b. Dilution 2: Add 1.0 mL of Dilution 1 to 9.0 mL

of LB broth. Cap and mix by inverting.

c. Dilution 3: Add 1.0 mL of Dilution 2 to 9.0 mL

of LB broth. Cap and mix by inverting.
C5. Add the calculated amount of 50 mg/mL streptomycin d. Dilution 4: Add 1.0 mL of Dilution 3 to 9.0 mL
to enough LB broth to result in a final solution volume of of LB broth. Cap and mix by inverting.
612 mL.
e. Dilution 5: Add 1.0 mL of Dilution 4 to 9.0 mL
C6. Bring the mixture of LB broth and streptomycin of LB broth. Cap and mix by inverting.
to 37 ˚C.
f. Dilution 6: Add 1.0 mL of Dilution 5 to 9.0 mL
C7. Measure the blank/baseline for the experiment using of LB broth. Cap and mix by inverting.
the UV-Visible spectrophotometer:
C15. Set up a Bunsen burner or alcohol lamp.
a. Fill a 1.0 cm cuvette with 2 mL of the warmed
LB broth. C16. For each time point, pipet 100 μL of Dilution 4, 5 and 6
onto separate plates.
b. Place the filled cuvette in the spectrophotometer
cuvette holder. C17. Sterilize a metal spreader by running it over a flame.
Allow to cool.
c. Close the sample compartment and press the
“Blank” button. C18. Use the sterilized or single-use spreader to spread the
solution across the entire surface of the agar plate in a
d. Remove the cuvette and dispose of the solution and circular motion.
cuvette in the appropriate waste disposal receptacles.
C19. Re-sterilize the spreader, if applicable, and repeat for
C8. Prepare the batch culture by transferring 12 mL of the each plate.
overnight culture to the warmed broth. Keep the
C20. Sterilize the spreader, if applicable, once again and allow
batch culture held at 37 ˚C and shaking at 300 rpm for
to cool before putting away.
the remainder of the experiment.
C21. Wait a few minutes for the sample to diffuse in the agar,
C9. Remove 5 mL from the batch culture using a
then incubate each plate overnight at 37 ˚C. Like in Part A,
sterile pipette.
the plates should be placed upside down in the incubator.

Measure
Collect 5 mL Add 2 mL to absorbance at
Aliquot 1.0 cm cuvette 600 nm

Repeat every
20 min.

Prepare batch culture

Figure 3. General OD 600 procedure from Part C. Created with BioRender.com.

 Lab Report
Viable colony
counting
Results Tables

Absorbance Absorbance
Growth Growth
at 600 nm at 600 nm
Time (min) Time (min)
(A.U.) (A.U.)
1. 1:10 Serial Dilutions

2. Spread plates with 100 μL

3. Incubate at 37 °C overnight to grow colonies

Figure 4. Viable cell counting procedure. Created with

BioRender.com.

Part D – Viable Colony Counting

D1. Using the plates spread from Part C, count the number
of viable colonies in each plate which can be counted
for each time point. It can be helpful to use a marker to
count each colony on the bottom of the plate to avoid Table 1. OD₆₀₀ Growth curve data.
re-counting errors. Consult your instructor for other
helpful methods for keeping track of the colony number
(e.g., count in batches, use a handheld counter, etc.) Growth Time Colony Count Colony Count
(min.) – Dilution – Stock
a. If the plate has too many colonies, use a marker to
divide the plates into quadrants. Count one quadrant
and multiply by 4 to estimate the total number of 0
colonies on the plate (See Figure 4).

D2. Back-calculate how many colonies were present in

60
the diluted solution for each countable plate. Record your
results in Table 2.

D3. Determine the bacterial cell count for each time point
120
in CFU/mL.

180

Table 2. Viable bacterial colony count data.

Based on the data in Table 1 and 2, construct the growth

curve for the samples analyzed herein. Include appropriate
units, axes labels and significant figures in the graph. Attach
the graph to the lab report questions. Include labels for the
bacterial growth phases observed.
Questions
1. From the data collected in this experiment, what phases of growth were you able
to observe?

2. What was the doubling time for this strain of E. coli calculated using the viable
colony counting data, and what was it when calculated using the OD₆₀₀ data?
Use the equation below for your analysis:

ln(2)(t₂ – t₁)
tD =
ln ( B₂
B₁
)
NOTE: Only the data collected in the exponential/log phase will be used for this analysis.

3. From Part A, what did your negative control look like? Why did we need to make a
negative control when streaking plates?

4. Why is it important the agar plates and LB broth have streptomycin?

5. What would you expect to happen to the growth curve if you added 24 mL of the
overnight culture instead of 12 mL to the LB broth?

6. What would you expect your growth curve to look like if you incubated the batch
culture at 25 ˚C instead of 37 ˚C?
Notes for Instructors
and Teaching Assistants:
Below includes an example of on OD₆₀₀ measurement coupled
with a viable cell count for an E. coli (strain K12) growth
experiment according to the procedure outlined in this lesson
plan with the exception of the intervals at which the aliquots
were measured. Note, the experimental procedure requires
5 hours to begin observing the end of the exponential/log
phase. If your lab period only lasts 3 hours, consider having
students compare their data to the figure below or complete
the experiment in your lab for the full 5 hours to produce a data
set for students to compare their data against.

1.5
Bacterial Colony Count (CFU/mL)

Viable Colony Count

10⁹ OD 600 Data
1.0
A₆₀₀

0.5

10⁸
0.0

0 50 100 150 200 250 300

Time (min)

Figure 5. Example OD 600 curve. The left axis correlates to the

black data points (bacterial colony count) and is plotted on
a logarithmic scale. The right axis represents the measured
absorbance at 600 nm and correlates to the red data points. Both
right and left axes were scaled to overlay well on one another.

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