Is Your Bacterial Culture Still Growing? A Primer on OD­600 Measurements

Learn the four stages of bacterial growth: lag phase (cells adapting), log/exponential phase (rapid cell division), stationary phase (maximum cell density maintained), and death phase (cell population decline). This foundational knowledge is essential for interpreting OD600 measurements.

Understand that OD600 measures optical density at 600 nanometers by quantifying light scattering caused by bacteria in culture. This wavelength is safe for cultures and is not absorbed by common media like TSB and LB broth.

Zero the spectrophotometer with fresh uninoculated medium, then measure OD600 of your culture sample immediately after thorough mixing. Avoid high OD readings (>1.0) by diluting samples as needed, and account for biofilms or aggregates that may affect accuracy.

Construct an OD600 calibration curve by preparing serial dilutions of your culture (ODs of 2, 1, 0.8, 0.6, 0.4, 0.2, and 0.1) and performing colony counts on plate dilutions. Use the linear relationship between OD and cells per milliliter to establish a conversion factor specific to your spectrophotometer and bacterial strain.

Train yourself to visually estimate OD in flasks to save time by skipping measurements when cultures are clearly outside your target range. Consider using a microplate reader for higher throughput of multiple OD600 measurements.

Employ supplementary techniques such as microscopy to assess cell viability and morphology, dissolved oxygen/CO2 monitoring in bioreactors, pH measurement during growth, or sugar analysis to determine where your culture is in the growth curve.