1. Collection of Bacterial Culture Aliquots

1. One day before collection of growth time points, inoculate 20 mL of trypticase soy broth (TSB) medium in a 50-mL flask with E. coli.
2. Incubate overnight at 27 °C. This relatively long incubation period results in a stationary phase population of wild type E. coli of approximately 10⁹ CFU/mL.
3. On the following day, use 100 µL of the prepared culture to inoculate 250 mL of TSB (in a 500-mL flask). Mix thoroughly.  Remove a 5-mL aliquot and refrigerate immediately at 4 °C. This is T = 0 or T₀ time point, and should contain approximately 4 x 10⁵ CFU/mL.
4. Place the flask of the remaining E. coli (245 mL) in a 37 °C shaking incubator. Remove 5-mL aliquots of culture every hour, up to 8 h. Store each aliquot at 4 °C. Designate these aliquots T₁ through T₈.

2. Serial Dilution

1. Remove aliquots of E. coli from the refrigerator and place on ice for transporting. Keep all cultures on ice until use.
2. Set up a series of dilution tubes to obtain various dilutions of each E. coli culture (Figure 3). Microfuge tubes are convenient for this function. Each dilution tube should have 900 µL of dilution fluid (sterile saline). A dilution series is needed for each E. coli culture (T₀ through T₈); label each tube according to Table 2.
   
   Figure 3. Schematic showing the procedure for plating E. coli.
3. Begin dilutions by adding 100 µL of E. coli from the tube labeled T₀ (the initial E. coli culture) to tube A, which is the 10^-1 dilution of T₀. Vortex the tube for 5 s.
4. Subsequently, add 100 µL of Tube A to the next tube of saline, Tube B, which is the 10^-2 dilution of T₀. Continue the needed dilution series for each time point aliquot. Remember to vortex each tube prior to transfer. It is also important to use a new pipette tip for each transfer.

Table 2. Dilution series required for each E. coli culture.

3. Plating

1. Three dilutions for each E. coli culture time point aliquot will be plated, according to Table 3. Label plates with the time point (T₁ through T₈), the dilution factor, and volume to be added. Use triplicate plates for each dilution.
2. Add 100 µL of each dilution to the plate by pipetting the amount to the center of the agar plate (Figure 3). Immediately spread the aliquot by utilizing a flame sterilized “L” shaped glass rod. If the aliquot is not spread immediately, it sorbs in situ on the plate, resulting in bacterial overgrowth at the spot of initial inoculation.
3. Repeat the plating for each dilution series for time points T₁ through T₈. Remember to sterilize the rod between plates and especially between different dilutions.
4. Once plates have dried for a few minutes, invert and place in 37 °C incubator overnight. Inverting the plates preclude condensation from falling onto the agar plate. Following overnight incubation, plates should be stored in refrigerator.

^*Lower dilutions take into account lower populations due to death phase.

Table 3. Plating protocol for E. coli cultures.

4. Counting Colonies and Calculating Mean Generation Time

1. Examine plates for uniformity of colonies and lack of contamination.
2. For each time point (T₀ through T₈), pick one dilution that contains between 30 and 300 colonies, and count triplicate plates.
3. Using the dilution factor, back-calculate the number of cells per mL of original culture at time points T₀ through T₈. For example, if the number of colonies resulting from a 10^-4 dilution is 30, 28, and 32:
   Mean number of colonies = 30 colonies
   These arose from 0.1 mL of a 10^-4 dilution
   Number of colonies per mL = 30 x 10⁴ = 3 x 10⁶
4. Plot log₁₀ CFU/mL versus time (in hours).
5. From the graph, identify the exponential phase of growth. Using 2 time points within the exponential growth phase and the corresponding cell numbers at each time, calculate the mean generation time.