OD600

OD600 refers to optical density measured at a wavelength of 600 nanometers and is widely used as a quick proxy for microbial culture density. The measurement relies on light scattering by cells in suspension; more cells scatter more light and transmit less, resulting in a higher OD600 reading. In many setups, OD600 is approximately proportional to cell concentration within a linear range, though the exact relationship depends on organism, cell size and shape, suspension quality, and the instrument’s path length.

Measurements are made with a spectrophotometer or plate reader. The culture is typically well mixed and the instrument is blanked with the medium or broth used to grow the culture. Path length matters: cuvettes commonly provide a 1 cm path length, while microplate wells have shorter and variable paths, requiring calibration or longer path-length corrections for accurate comparisons.

OD600 should be used within the instrument’s linear range, often around 0.1 to 0.8 for bacteria using cuvettes. Dilution may be necessary for denser cultures. While convenient for tracking growth and standardizing inocula, OD600 is not a direct measure of viable cells and can be influenced by cell pigmentation, clumping, or debris. Therefore, species-specific standard curves relating OD600 to cells per milliliter are recommended for quantitative estimates, and alternative methods like CFU counts or direct cell enumeration should be used for precise viability or concentration assessments. OD600 remains a staple for rapid, non-destructive growth monitoring in microbiology.