Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements
1. Set-up
- Required laboratory materials: liquid media, solidified agar media, Erlenmeyer flasks, 15 mL test tubes, phosphate buffered saline (PBS), bacterial cell spreader, 70% ethanol, and a spectrophotometer. All solutions and glassware must be sterilized prior to use.
- Prepare the work station by sterilizing with 70% ethanol. Work near a Bunsen burner to prevent contamination of media.
- When working with bacteria, proper personal protective equipment and aseptic technique should be used. A lab coat and gloves are required when working with bacterial cultures.
- Recipes for buffers, solutions, and reagents
- Phosphate buffered saline (PBS) (8).
- Luria-Bertani Broth (LB) (9).
2. Protocol
- Preparation of Media
- Identify the growth media with which to grow the bacteria and prepare both liquid broth and solid agar (1.5% w/v agar) media in separate autoclavable bottles. Here, LB broth and LB agar were prepared for the growth of Escherichia coli.
- Sterilize the media with a semi-tightened cap in an autoclave set to 121 °C for 35 min.
- For agar media, after autoclaving, place in a water bath set to 50 °C for 30 minutes to cool. Once cooled, pour 20-25 mL agar media into 100x15mm circular Petri dishes. Allow plates to set 24 hours at room temperature before use.
- Initial Preparation of Bacteria
- From frozen stock, streak bacteria for isolation on selected media agar to obtain single colony isolates. Incubate in growth conditions permissible for the chosen bacteria. Here, E. coli is streaked on LB agar and is incubated at 37 °C overnight (16-18h).
- Using a sterile inoculation loop, select a single colony from the streak plate and inoculate 4 mL liquid media in a 15 mL test tube and grow in conditions permissible for the chosen bacteria. Here, E. coli is grown at 37 °C with shaking at 210 rpm overnight (16-18h).
- Growth Curve Setup
- Growth flask preparation
- Autoclave appropriately sized Erlenmeyer flasks. Typically, a 1:5 ratio of media to total flask volume is used. Here, 100 mL LB media is used in a 500 mL flask.
- Using a serological pipette, transfer sterile media to the Erlenmeyer flask.
- Dilution series preparation
- Label 15 mL test tubes: -1, -2, -3, -4, -5, -6, -7, -8, and -9, distributing 9mL PBS into each. These numbers correspond to the dilution factor used to calculate CFU/mL. A new set of tubes is needed for each collection timepoint. (Figure 2)
- Agar plate preparation
- Label plates with time of collection and dilution factor. For each timepoint there will be one plate for each dilution.
- Growth flask preparation
- Growth Curve Protocol
- Inoculation of media
- Using the overnight liquid culture prepared as part of step 2.2.2, inoculate the flask media with 1:1000 volume of culture. Here, 100 µL overnight liquid culture is added to 100 mL LB media.
- Swirl the media to evenly distribute the bacteria.
- Timepoint collection
- Growth condition setup
- Place flask in experimental growth conditions chosen for the given bacteria. Timepoints should be taken frequently for fast-growing bacteria and can be taken in longer intervals for slow-growing bacteria. Here, E. coli is grown at 37°C with shaking at 210 revolutions per minute (rpm) and timepoints taken every 1 hour.
- Optical density (OD600) measurement
- At each timepoint, including the starting timepoint (t = 0), withdraw 1 mL of bacterial culture and dispense into a spectrophotometer cuvette.
- Wipe the cuvette clean and record the optical density at 600 nm wavelength. If the optical density reading is greater than 1.0, dilute 100 µL of culture 1:10 with 900 µL fresh media, record the optical density, and multiply this value by 10 for the OD600 measurement.
- Colony forming unit (CFU/mL) measurement
- At each timepoint, withdraw 1 mL of bacterial culture and dispense into the -1 glass test tube containing 9 mL of PBS.
- For the dilution series, serially transfer 1 mL from the -1 tube down all dilution tubes to the -9, vortexing after each transfer. (Figure 2)
- For each dilution, dispense 100 µL of cell suspension to the correspondingly labelled solid media agar plate. (Figure 2)
- Using a cell spreader that has been sterilized in ethanol, passed through a Bunsen burner flame, and cooled by touching the surface of the agar, spread the 100 µL of cell suspension until the surface of the agar plate becomes dry.
- Incubate the spread plates upside-down at a temperature that supports growth of the bacteria. Here, E. coli is incubated at 37°C.
- After incubation, once visible colonies arise, count the number of bacterial colonies on each plate and record these values along with their associated dilution factor for all plates at each timepoint.
- Growth condition setup
- Inoculation of media
3. Data Analysis and Results
- Optical Density (OD600) Growth Curve Plot
- Plot the optical density (OD600) versus time on a semi-log scale. (Figure 3)
- Colony Forming Unit (CFU/mL) Growth Curve Plot
- For each timepoint, choose the dilution plate where the colony counts fell within the range of 30-300 bacteria. Multiply the colony count number by the dilution factor and then by 10 as the 100 µL spread is considered an additional 1:10 dilution when calculating CFU/mL.
- Plot the colony forming units versus time on a semi-log scale. (Figure 4)
- For each timepoint, choose the dilution plate where the colony counts fell within the range of 30-300 bacteria. Multiply the colony count number by the dilution factor and then by 10 as the 100 µL spread is considered an additional 1:10 dilution when calculating CFU/mL.
- Relating optical density and colony forming units
- Plot the colony forming units versus optical density on a linear scale for OD600 readings less than or equal to 1.0 OD600 as the relationship between OD600 and CFU/mL is less accurate beyond 1.0 OD600. Here, the first six timepoints are plotted. (Figure 5)
- Generate a linear regression trendline displaying the equation and R2 value.
- Determining bacteria doubling time
- Using the colony forming unit growth curve plot, during exponential phase, identify two points on the graph with the steepest slope between them to calculate the doubling time.
- Calculating the doubling time
- ΔTime = t2 - t1, where t1 = Timepoint 1 and t2 = Timepoint 2
, where b = number of bacteria at t2, B = number of bacteria at t1, and n = number of generations. Derived from: .
- Calculate doubling time using:
Source: Andrew J. Van Alst1, Rhiannon M. LeVeque1, Natalia Martin1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michiga…
1. Set-up
- Required laboratory materials: liquid media, solidified agar media, Erlenmeyer flasks, 15 mL test tubes, phosphate buffered saline (PBS), bacterial cell spreader, 70% ethanol, and a spectrophotometer. All solutions and glassware must be sterilized prior to use.
- Prepare the work station by sterilizing with 70% ethanol. Work near a Bunsen burner to prevent contamination of media.
- When working with bacteria, proper personal protective equipment and aseptic technique should be used. A lab coat and gloves are required when working with bacterial cultures.
- Recipes for buffers, solutions, and reagents
- Phosphate buffered saline (PBS) (8).
- Luria-Bertani Broth (LB) (9).
2. Protocol
- Preparation of Media
- Identify the growth media with which to grow the bacteria and prepare both liquid broth and solid agar (1.5% w/v agar) media in separate autoclavable bottles. Here, LB broth and LB agar were prepared for the growth of Escherichia coli.
- Sterilize the media with a semi-tightened cap in an autoclave set to 121 °C for 35 min.
- For agar media, after autoclaving, place in a water bath set to 50 °C for 30 minutes to cool. Once cooled, pour 20-25 mL agar media into 100x15mm circular Petri dishes. Allow plates to set 24 hours at room temperature before use.
- Initial Preparation of Bacteria
- From frozen stock, streak bacteria for isolation on selected media agar to obtain single colony isolates. Incubate in growth conditions permissible for the chosen bacteria. Here, E. coli is streaked on LB agar and is incubated at 37 °C overnight (16-18h).
- Using a sterile inoculation loop, select a single colony from the streak plate and inoculate 4 mL liquid media in a 15 mL test tube and grow in conditions permissible for the chosen bacteria. Here, E. coli is grown at 37 °C with shaking at 210 rpm overnight (16-18h).
- Growth Curve Setup
- Growth flask preparation
- Autoclave appropriately sized Erlenmeyer flasks. Typically, a 1:5 ratio of media to total flask volume is used. Here, 100 mL LB media is used in a 500 mL flask.
- Using a serological pipette, transfer sterile media to the Erlenmeyer flask.
- Dilution series preparation
- Label 15 mL test tubes: -1, -2, -3, -4, -5, -6, -7, -8, and -9, distributing 9mL PBS into each. These numbers correspond to the dilution factor used to calculate CFU/mL. A new set of tubes is needed for each collection timepoint. (Figure 2)
- Agar plate preparation
- Label plates with time of collection and dilution factor. For each timepoint there will be one plate for each dilution.
- Growth flask preparation
- Growth Curve Protocol
- Inoculation of media
- Using the overnight liquid culture prepared as part of step 2.2.2, inoculate the flask media with 1:1000 volume of culture. Here, 100 µL overnight liquid culture is added to 100 mL LB media.
- Swirl the media to evenly distribute the bacteria.
- Timepoint collection
- Growth condition setup
- Place flask in experimental growth conditions chosen for the given bacteria. Timepoints should be taken frequently for fast-growing bacteria and can be taken in longer intervals for slow-growing bacteria. Here, E. coli is grown at 37°C with shaking at 210 revolutions per minute (rpm) and timepoints taken every 1 hour.
- Optical density (OD600) measurement
- At each timepoint, including the starting timepoint (t = 0), withdraw 1 mL of bacterial culture and dispense into a spectrophotometer cuvette.
- Wipe the cuvette clean and record the optical density at 600 nm wavelength. If the optical density reading is greater than 1.0, dilute 100 µL of culture 1:10 with 900 µL fresh media, record the optical density, and multiply this value by 10 for the OD600 measurement.
- Colony forming unit (CFU/mL) measurement
- At each timepoint, withdraw 1 mL of bacterial culture and dispense into the -1 glass test tube containing 9 mL of PBS.
- For the dilution series, serially transfer 1 mL from the -1 tube down all dilution tubes to the -9, vortexing after each transfer. (Figure 2)
- For each dilution, dispense 100 µL of cell suspension to the correspondingly labelled solid media agar plate. (Figure 2)
- Using a cell spreader that has been sterilized in ethanol, passed through a Bunsen burner flame, and cooled by touching the surface of the agar, spread the 100 µL of cell suspension until the surface of the agar plate becomes dry.
- Incubate the spread plates upside-down at a temperature that supports growth of the bacteria. Here, E. coli is incubated at 37°C.
- After incubation, once visible colonies arise, count the number of bacterial colonies on each plate and record these values along with their associated dilution factor for all plates at each timepoint.
- Growth condition setup
- Inoculation of media
3. Data Analysis and Results
- Optical Density (OD600) Growth Curve Plot
- Plot the optical density (OD600) versus time on a semi-log scale. (Figure 3)
- Colony Forming Unit (CFU/mL) Growth Curve Plot
- For each timepoint, choose the dilution plate where the colony counts fell within the range of 30-300 bacteria. Multiply the colony count number by the dilution factor and then by 10 as the 100 µL spread is considered an additional 1:10 dilution when calculating CFU/mL.
- Plot the colony forming units versus time on a semi-log scale. (Figure 4)
- For each timepoint, choose the dilution plate where the colony counts fell within the range of 30-300 bacteria. Multiply the colony count number by the dilution factor and then by 10 as the 100 µL spread is considered an additional 1:10 dilution when calculating CFU/mL.
- Relating optical density and colony forming units
- Plot the colony forming units versus optical density on a linear scale for OD600 readings less than or equal to 1.0 OD600 as the relationship between OD600 and CFU/mL is less accurate beyond 1.0 OD600. Here, the first six timepoints are plotted. (Figure 5)
- Generate a linear regression trendline displaying the equation and R2 value.
- Determining bacteria doubling time
- Using the colony forming unit growth curve plot, during exponential phase, identify two points on the graph with the steepest slope between them to calculate the doubling time.
- Calculating the doubling time
- ΔTime = t2 - t1, where t1 = Timepoint 1 and t2 = Timepoint 2
- , where b = number of bacteria at t2, B = number of bacteria at t1, and n = number of generations. Derived from:
.
- Calculate doubling time using:
Bacteria reproduce through a process called cell division, which results in two identical daughter cells. If the growth conditions are favorable, bacterial populations will grow exponentially.
Bacterial growth curves plot the amount of bacteria in a culture as a function of time. A typical growth curve progresses through four stages: lag phase, exponential phase, stationary phase, and death phase. The lag phase is the time it takes for bacteria to reach a state where they can grow and divide quickly. After this, the bacteria transition to the exponential phase, characterized by rapid cell growth and division. The rate of exponential growth of the bacterial culture during this phase can be expressed as the doubling time, the fastest rate at which bacteria can reproduce under specific conditions. The stationary phase comes next, where bacterial cell growth plateaus and the growth and death rates even out due to environmental nutrient depletion. Finally, the bacteria enter the death phase. This is where bacterial growth declines sharply and severe nutrient depletion leads to the lysing of cells.
Two techniques can be used to quantify the amount of bacteria present in a culture and plot a growth curve. The first of these is via colony forming units, or CFUs. To obtain CFUs a one to ten series of nine dilutions is performed at regular time points. The first of these dilutions, negative one in this example, contains 9mL of PBS and 1mL of the bacterial culture. Resulting in a 1:10 dilution factor. Then, 1mL of this solution is transferred to the next tube, negative two, resulting in a 1:100 dilution factor. This process continues through the last tube, negative nine, resulting in a final dilution factor of 1:1 billion. After this, 100 microliters of each dilution is plated. The plates are then incubated and the clonal colonies are counted. The dilution plate for a given time point that grows between 30 and 300 colonies is used to calculate the CFUs per milliliter for that time point.
The second common method of measuring bacterial concentration is the optical density. The optical density of a culture can be measured instantly, in relation a media blank, with a spectrophotometer. Typically a wave length of 600 nanometers, also referred to as OD600, is used for these measurements, which increase as cell density increases. While optical density is less precise than CFUs, it is convenient because it can be obtained instantaneously and requires relatively few reagents. Both techniques can be used together to create a standard curve that more accurately approximates the bacterial cell count of a culture. In this video, you will learn how to obtain CFUs and OD600 measurements from timed serial dilutions of E. coli. Then, two growth curves using the CFU and OD600 measurements, respectively, will be plotted before being related by a standard curve.
When working with bacteria, it is important to use the appropriate personal protective equipment such as a lab coat and gloves and to observe proper aseptic technique.
After this, sterilize the work station with 70% ethanol. First, prepare the LB broth and LB solid agar media in separate autoclaveable bottles. After partially closing the caps of the bottles, sterilize the media in an autoclave set to 121 degrees Celsius for 35 minutes. Next, allow the agar media to cool in a water bath set to 50 degrees Celsius for 30 minutes. Once cooled, pour 20 to 25 mL into each Petri dish. After this, allow the plates to set for 24 hours at room temperature.
To prepare the single colony isolates that will later be used to produce a liquid bacterial culture, use previously frozen stock and proper streak plating technique to streak E. coli for isolation on LB agar. Incubate the dish at 37 degree Celsius overnight. After this, cool a flame sterilized inoculation loop on the agar before selecting a single colony from the streaked plate. Inoculate 4 mL of liquid media in a 15 mL test tube. Then, grow the E. coli at 37 degrees Celsius overnight with shaking at 210 rpm.
To set up the 1:1000 volume of bacterial culture that will be used in the growth curve, first obtain an autoclaved 500 mL Erlenmeyer flask. Then, use a 50 mL serological pipette to transfer 100 mL of sterile media to the flask. Next, label nine 15 ml test tubes consecutively as one through nine. These numbers correspond to the dilution factor that will be used to calculate the colony forming unit, or CFU. Then, add 9 mL of 1X PBS to each tube. After this, label the prepared agar plates with the corresponding time points and dilution factors that will be grown. In this example with E. coli, after the starting time point, time points are taken once every hour. Using the previously prepared overnight liquid E. coli culture, inoculate the media in the autoclave 500 mL Erlenmeyer flask with 1:1000 volume of culture. Swirl the media to evenly distribute the bacteria.
After blanking a spectrophotometer, clean the cuvette with a lint-free wipe. Next, dispense 1 mL of the culture into the cuvette and place it into the spectrophotometer to obtain the optical density of the culture at time point zero. Then, grow the E. coli at 37 degrees Celsius with shaking at 210 rpm. At each time point after time point zero, withdraw another 1 mL of bacterial culture from the flask and repeat the optical density measurement. If the optical density reading is greater than 1.0, dilute 100 microliters of bacterial culture with 900 microliters of fresh media and then measure the optical density once more. This value can be multiplied by 10 for the OD 600 measurement.
To obtain the colony forming unit measurement for each time point, withdraw an additional 1 mL of bacterial culture from the flask at each time point. Dispense the bacterial culture into the negative one test tube and vortex to mix. Then, perform the dilution series by first transferring 1 mL from the negative one tube into the negative two tube and vortex to mix. Transfer 1 mL from the negative two tube into the negative three tube and vortex to mix. Continue this serial transfer down all the dilution tubes to the negative nine tube. Dispense 100 microliters of cell suspension onto the correspondingly labeled plate for each dilution. For every dilution, sterilize a cell spreader in ethanol, pass it through a Bunsen burner flame, and cool it by touching the surface of the agar away from the inoculate. Then, use the cell spreader to spread the cell suspension until the surface of the agar plate becomes dry. Incubate the plates upside down at 37 degrees Celsius. Once visible colonies arise, count the number of bacterial colonies on each plate. Record these values and their associated dilution factors for each plate at each time point.
To create an OD 600 growth curve, after ensuring all the data points are entered correctly into a table, select all of the time points and their corresponding data. To generate a colony forming unit growth curve plot, choose the dilution plate where the colony counts fell within the range 30 to 300 bacteria for each time point. Multiply the colony count number by the dilution factor, and then by ten. This is because the 100 microliters spread is considered an additional 1:10 dilution when calculating colony forming units per milliliter. After this, plot the colony forming units versus time on a semi-log scale.
These plots produced with OD 600 and CFU measurements, respectively, can provide valuable information on E. coli growth kinetics. The optical density and colony forming units can be related, so that CFUs per milliliter can be estimated from OD 600 measurements, saving time and materials in future experiments.
To do this, plot the colony forming units against the optical density on a linear scale for OD 600 readings less than or equal to 1. 0. After this, generate a linear regression trend line in Y = MX + B format, where M is the slope and B is the y-intercept. Right click on the data points and select add trend line and linear. Then, check the box to display the equation on the chart and display the R squared value on the chart. The R squared value is the statistical measurement of how closely the data matched the fitted regression line. In this example, the first 6 time points are plotted with OD 600 on the x axis and CFUs per milliliter on the y axis. In future experiments with the same growth conditions, these slope and y-intercept values can be plugged into this equation to estimate CFUs from OD 600 readings. Next, look at the colony forming unit growth curve plot. During the exponential phase, identify two time points with the steepest slope between them. To calculate the doubling time, first calculate the change in time between the selected time points. Then, calculate the change in generations using the equation shown here. Here, lower case b is the number of bacteria at time point three and upper case B is the number of bacteria at time point two. Finally, divide the change in time by the change in generations. In this example, the doubling time is 0. 26 hours or 15 minutes and 19 seconds. Comparing doubling times across different experimental treatment allows us to identify the best growth conditions for a certain bacterial species. Therefore, the treatment with the lowest doubling time will be most optimal of the conditions tested.
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Q1: What are the four phases of a bacterial growth curve?
A typical bacterial growth curve progresses through four distinct phases. Lag phase is when bacteria adjust to their environment before rapid growth begins. Exponential phase follows, characterized by rapid cell division and doubling. Stationary phase occurs when growth plateaus due to nutrient depletion. Finally, death phase involves cell lysis from severe nutrient limitation.
Q2: How do colony forming units differ from optical density measurements?
Colony forming units (CFUs) provide direct bacterial cell counts through serial dilution and plate counting, yielding precise CFU/mL values. Optical density measures light absorbance at 600 nanometers (OD600) using a spectrophotometer, offering instant results with fewer reagents. While OD600 is faster and more convenient, CFUs are more accurate, especially at higher cell densities.
Q3: Why is the 30-300 colony range important when counting plates?
The 30-300 colony range minimizes counting error when calculating CFU/mL. Plates with fewer than 30 colonies show high stochastic variation, leading to unreliable estimates. Plates exceeding 300 colonies suffer from crowding and overlapping, causing underestimation. Using plates within this optimal range ensures accurate bacterial enumeration for each time point.
Q4: What is doubling time and how is it calculated?
Doubling time is the fastest rate at which bacteria reproduce under specific growth conditions, measured during the exponential phase. To calculate it, identify two time points with the steepest slope, then compute the change in time and change in generations using the bacterial counts at each point. Dividing time change by generation change yields doubling time, allowing comparison of growth conditions.
Q5: How does a standard curve relate optical density to colony forming units?
A standard curve plots CFU/mL against OD600 on a linear scale for readings at or below 1.0 OD600. A linear regression equation (Y = MX + B) is generated, where M is the slope and B is the y-intercept. This relationship allows researchers to estimate CFU/mL from OD600 measurements in future experiments, saving time and materials while maintaining accuracy.
Q6: What preparation steps are necessary before measuring bacterial growth?
First, prepare LB broth and agar media in separate bottles, then sterilize in an autoclave at 121°C for 35 minutes. Cool agar to 50°C and pour into Petri dishes. Using pure cultures and streak plating isolation of single bacterial colonies, inoculate liquid media overnight at 37°C with shaking. Finally, inoculate a sterile flask with a 1:1000 volume of the overnight culture to begin growth curve measurements.
Q7: Why does the relationship between OD600 and CFU/mL deteriorate at higher optical densities?
The OD600-to-CFU relationship becomes less accurate above 1.0 OD600 because bacterial cells begin changing shape and accumulating extracellular products in the media. These changes influence light absorbance readings independently of actual cell count, causing the spectrophotometer measurement to diverge from true bacterial enumeration. This error becomes pronounced during stationary and death phases.
Chapters in this video
0:01
Concepts
3:44
Set-Up
8:52
Data Analysis and Results
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