Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements

1. Set-up

1. 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.
2. Prepare the work station by sterilizing with 70% ethanol. Work near a Bunsen burner to prevent contamination of media.
3. 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.
4. Recipes for buffers, solutions, and reagents
   1. Phosphate buffered saline (PBS) (8).
   2. Luria-Bertani Broth (LB) (9).

2. Protocol

1. Preparation of Media
   1. 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.
   2. Sterilize the media with a semi-tightened cap in an autoclave set to 121 °C for 35 min.
   3. 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.
2. Initial Preparation of Bacteria
   1. 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).
   2. 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).
3. Growth Curve Setup
   1. Growth flask preparation
      1. 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.
      2. Using a serological pipette, transfer sterile media to the Erlenmeyer flask.
   2. Dilution series preparation
      1. 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)
   3. Agar plate preparation
      1. Label plates with time of collection and dilution factor. For each timepoint there will be one plate for each dilution.
4. Growth Curve Protocol
   1. Inoculation of media
      1. 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.
      2. Swirl the media to evenly distribute the bacteria.
   2. Timepoint collection
      1. Growth condition setup
         1. 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.
      2. Optical density (OD600) measurement
         1. At each timepoint, including the starting timepoint (t = 0), withdraw 1 mL of bacterial culture and dispense into a spectrophotometer cuvette.
         2. 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.
      3. Colony forming unit (CFU/mL) measurement
         1. At each timepoint, withdraw 1 mL of bacterial culture and dispense into the -1 glass test tube containing 9 mL of PBS.
         2. 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)
         3. For each dilution, dispense 100 µL of cell suspension to the correspondingly labelled solid media agar plate. (Figure 2)
         4. 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.
         5. Incubate the spread plates upside-down at a temperature that supports growth of the bacteria. Here, E. coli is incubated at 37°C.
         6. 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.

3. Data Analysis and Results

1. Optical Density (OD600) Growth Curve Plot
   1. Plot the optical density (OD600) versus time on a semi-log scale. (Figure 3)
2. Colony Forming Unit (CFU/mL) Growth Curve Plot
   1. 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.
      
   2. Plot the colony forming units versus time on a semi-log scale. (Figure 4)
3. Relating optical density and colony forming units
   1. 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)
   2. Generate a linear regression trendline displaying the equation and R² value.
4. Determining bacteria doubling time
   1. 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.
   2. Calculating the doubling time
      1. 
      2. ΔTime = t₂ - t₁, where t₁ = Timepoint 1 and t₂ = Timepoint 2
      3. , where b = number of bacteria at t₂, B = number of bacteria at t₁, and n = number of generations. Derived from: .
      4. Calculate doubling time using: