What is OD600
Optical density at 600 nanometres (OD600) is the most common method for tracking bacterial cell concentration in liquid culture. A spectrophotometer passes light through a cuvette of culture broth and measures the fraction that reaches the detector. The number reported is called absorbance, but in a turbid bacterial suspension the signal is dominated by light scattering, not absorption.[1][2] More cells, larger cells, and more particles in the path all increase the reading.
The wavelength of 600 nm is a practical choice rather than a fundamental one. It sits in a window where most bacterial cultures scatter light strongly while pigments and common medium components absorb relatively little.[1] That tradeoff between sensitivity and chemical specificity is why 600 nm became the de facto standard, even though wavelengths from 540 nm to 660 nm are sometimes used.
OD600 is fast, cheap, and ubiquitous. Every cultivation lab has the equipment, every protocol quotes it, and every microbiologist knows the readout. What it gives you is a turbidity-based proxy for biomass — not a direct count of cells.
Applications of OD600
OD600 has two dominant uses in cultivation work.
- Growth curves. Repeated readings through a culture, taken every few minutes to every few hours depending on the organism, are used to build a growth curve. The curve is then read for the lag, exponential, stationary, and decline phases of bacterial growth.
- Culture standardisation. OD600 is used to dose bacteria into downstream experiments at a target density, ensuring that experimental replicates start with comparable cell concentrations. The same logic applies when seeding fermenters at a defined inoculum.
The reason OD600 is so widely used is operational: a measurement takes under a minute and costs nothing per sample beyond the cuvette. The downside is what the number actually represents.
Considerations and limitations
OD600 is a biomass-related signal, not a cell count, and the gap between the two has practical consequences.
OD does not distinguish cells from other particles. Insoluble medium components, antifoam droplets, precipitates after pH or temperature shifts, and cell debris from lysis all add to the reading.[3] OD cannot tell them apart from cells.
OD depends on cell size and morphology. Two cultures with identical cell numbers but different cell sizes will give different OD values. Stress, nutrient limitation, and approaching stationary phase all shift cell size, so OD can keep changing while the cell count is steady.[4][5]
OD does not distinguish cells by physiological state. Microbiology recognises a continuum of states between active growth and full lysis: cells that are dividing, cells that have stopped dividing but remain metabolically active, viable but non-culturable cells, cells with damaged membranes, and lysed debris.[6] OD600 measures turbidity, and any cell or fragment that remains structurally intact contributes to that signal regardless of which state it is in. After lysis, the released debris continues to contribute until it settles or is degraded. There is no single OD600 reading at which cells stop counting toward the signal.
OD600 reports an arbitrary number. Without a calibration curve specific to the organism and the instrument — and revalidated when the medium changes — OD600 is not convertible to cells/mL or any standard biological unit.[5][7]
OD600 has a narrow linear range. Above an absorbance of roughly half a unit in a standard 1 cm cuvette, the relationship between signal and concentration becomes non-linear, and dilution is required to stay in range.[4]
OD readings are instrument-dependent. Spectrophotometer geometry, detector design, and bandpass differ between models. Two instruments will give different OD values for the same sample unless they are cross-calibrated.[7]
Alternatives
Several methods coexist with OD600 in cultivation labs. Each answers a slightly different question.
| Method | What it measures | Strengths | Limitations |
|---|---|---|---|
| CFU plating | Cells competent to form colonies on a defined medium | Direct, biologically meaningful, regulatory standard for QC release | Slow (24–72 h or longer), labour-intensive, misses viable-but-non-culturable cells, undercounts aggregates |
| Dry cell weight | Total dried biomass per volume | Calibrated mass unit, used for yield calculations | Slow (hours), destructive, insensitive at low concentrations, biomass is not cell number |
| Fluorescence flow cytometry | Per-cell scatter and fluorescence; can include viability and metabolism dyes | Rich per-cell information, fast at the time-point | Expensive; complex sample prep; primarily designed for eukaryotes; undercounts aggregates; does not directly detect loss of culturability |
| BactoBox (impedance flow cytometry) | Concentration of structurally intact total bacterial cells (cells/mL) | Direct bacterial cell count in ~2 minutes per sample, benchtop format, clean interpretable data without interference | Electrical signal only — no versatile tags or dyes; undercounts aggregates; does not directly detect loss of culturability |
Common behavior of OD600
Common questions and answers.
Why does my OD600 keep rising after my cells have stopped dividing?
Cells continue to change size and refractive properties for a while after division has slowed or stopped. Depending on the organism and the limitation that triggered slowdown, cells may shrink, swell with storage granules such as polyhydroxyalkanoates or glycogen, or change shape.[8][9] Each of those changes alters how the cell scatters light at 600 nm even though no new cells have been added.[1][4] The result is an OD600 that drifts upward — or downward — even though the cell count has plateaued. The OD plateau and the moment cell division actually ends rarely line up exactly.
Can OD600 tell me whether my cells are alive or dead?
No, but the more accurate question is what definition of "alive" the experiment requires, because microbiology recognises several distinct definitions and OD600 cannot answer any of them.[6] Cells can be actively dividing, non-dividing but metabolically active, viable but non-culturable, structurally intact but metabolically inactive, or partially lysed. Each of these states still contributes to turbidity to varying degrees, because turbidity reflects the bulk presence of light-scattering material in the broth, not any individual cell's state. A flat or slowly declining OD tail can therefore hide a culture that has lost culturability without losing structure, a population of intact cells that is dropping, or a culture beginning to lyse. Asking whether cells are alive requires deciding which definition matters for the decision at hand — culturable, metabolically active, structurally intact, or membrane-intact — and choosing a method that targets that specific definition.
Can I use OD600 to compare different media, strains, or process conditions?
The entire industry does, not without success, but OD600 is not the best method for this. Even after blanking the medium, OD600 still depends on cell size and intracellular composition — and both of those properties are themselves shaped by the medium, the strain, and the cultivation conditions a cell grows under. A medium that produces cells with denser cytoplasm or more storage compounds reads higher than a medium producing the same cell count with leaner cells. Two strains with different cell sizes will give different OD curves at the same true cell concentration. Blanking corrects for the optics of the broth itself; it does not correct for what the broth has done to the cells. There is no single calibration factor that survives a change in medium or strain, because the OD-to-cell ratio depends on cell properties that the new condition has changed. The error is silent: the OD curve looks clean, the ranking is internally consistent, the result is reproducible — and the ranking can still be wrong, with no diagnostic from inside OD that tells you when. The only way to compare cell yields across media, strains, or process conditions reliably is to count cells directly. We cover this in detail in our article on rational media selection.
Why does my OD600 jump after I add antifoam, inducer, or a base correction?
Many additions introduce particulates, precipitates, or refractive-index shifts that change the OD signal independently of the cell population. Antifoams form droplets that scatter light. pH corrections can precipitate medium components. Inducer stocks sometimes carry insolubles. The OD jump is real, but it is not a change in the cells.
Why is my OD600 different on a different spectrophotometer?
OD600 is not a standardised quantity. Instruments differ in optical path geometry, detector design, and spectral bandpass, so the same sample can read differently between machines. A 2020 inter-laboratory study across 244 laboratories found that calibration against serial dilutions of silica microspheres allows OD-derived cell counts to be compared across instruments, but without such calibration, OD readings cannot be meaningfully compared between instruments.[7]
Closing
OD600 remains fast and convenient, but it is not a cell count. For decisions that depend on knowing how many cells are in the broth — when to harvest, how strains compare, whether a run is reproducible — methods that count cells directly answer the question OD was never designed to answer.
References
- Myers JA, Curtis BS, Curtis WR. Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophys. 2013;6:4. https://doi.org/10.1186/2046-1682-6-4
- Koch AL. Turbidity measurements of bacterial cultures in some available commercial instruments. Anal Biochem. 1970;38(1):252-9. https://doi.org/10.1016/0003-2697(70)90174-0
- Routledge SJ. Beyond de-foaming: the effects of antifoams on bioprocess productivity. Comput Struct Biotechnol J. 2012;3:e201210014. https://doi.org/10.5936/csbj.201210014
- Stevenson K, McVey AF, Clark IBN, Swain PS, Pilizota T. General calibration of microbial growth in microplate readers. Sci Rep. 2016;6:38828. https://doi.org/10.1038/srep38828
- Mira P, Yeh P, Hall BG. Estimating microbial population data from optical density. PLoS One. 2022;17(10):e0276040. https://doi.org/10.1371/journal.pone.0276040
- Davey HM. Life, death, and in-between: meanings and methods in microbiology. Appl Environ Microbiol. 2011;77(16):5571-6. https://doi.org/10.1128/AEM.00744-11
- Beal J, Farny NG, Haddock-Angelli T, Rai V, Davies J, Patron N, et al. Robust estimation of bacterial cell count from optical density. Commun Biol. 2020;3:512. https://doi.org/10.1038/s42003-020-01127-5
- Åkerlund T, Nordström K, Bernander R. Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli. J Bacteriol. 1995;177(23):6791-7. https://doi.org/10.1128/jb.177.23.6791-6797.1995
- Slaninova E, Sedlacek P, Mravec F, Mullerova L, Samek O, Krzyzanek V, et al. Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation. Appl Microbiol Biotechnol. 2018;102(4):1923-31. https://doi.org/10.1007/s00253-018-8760-8