Figure 1.

Nominal growth curve and comparative day 2 growths on different media.A, nominal bacterial growth curve. Growth is monitored by the A₆₀₀, rather than the typical doubling time. A₆₀₀ from 0.2 to 1.0 is the preferred growth window. Cell density at initial inoculation and each expanding step is maintained at ~0.2 to 0.3 for healthy growth, time-saving, and fast amplification. B, a general timeline for day 2 cell culture in all three media. ²H-M9 culture goes through training as described in Figure 2. For ¹H-M9, a small 2 ml starter culture is grown during the day to reach A₆₀₀ ~0.2 to 0.3, then added into 100 ml media for overnight growth. For LB growth, inoculate a dozen colonies directly into 50 ml media for overnight. Note, day 2 overnight culture volumes are different for each media reflecting the cell density in each media after overnight growth, with LB at the highest, ²H-M9 at the lowest. By adjusting the volume accordingly, the initial cell density on day 3 is ensured to be around 0.2, thus maintaining the exponential growth window.