Cell density: OD₆₀₀, hemocytometer counts and seeding
Turn an optical density or a grid count into cells per millilitre, then work out what to pipette to hit a target density.
OD₆₀₀ → cells per mL
Conversion factors are strain-, phase- and instrument-dependent. Calibrate against your own plate counts for anything quantitative.
Hemocytometer count
Count the four (or five) large corner squares, average them, and enter the average here along with any dilution — including trypan blue.
Seeding and passaging
How much of your suspension goes into each well, and how much medium to add.
Growth rate and doubling time
From two density readings taken during exponential growth.
Growth phases and when to harvest
| Phase | Typical E. coli OD₆₀₀ | Use it for |
|---|---|---|
| Lag | < 0.05 | Nothing — let it recover |
| Early exponential | 0.1–0.3 | Competent-cell prep, growth-rate measurements |
| Mid exponential | 0.4–0.8 | IPTG induction, phage infection |
| Late exponential | 0.8–1.5 | Plasmid miniprep, biomass |
| Stationary | > 2 | Stress-response studies, secondary metabolites |
Why OD₆₀₀ is not really an absorbance
Cells do not absorb much at 600 nm — they scatter. The “absorbance” a spectrophotometer reports is light removed from the straight-through beam, which depends on cell size, shape, refractive index and on the geometry of the instrument's detector. Two spectrophotometers can differ by 30 % on the same sample, and a plate reader with a short path length will read lower still.
Consequences worth remembering:
- Beer's law holds only up to about OD 0.8. Above that, dilute into the linear range and multiply back — do not extrapolate.
- Cells elongate under stress, which raises OD without raising cell number.
- Published OD-to-cell-count factors apply to the instrument they were measured on. If you need real numbers, plate for colony counts and build your own calibration curve.
Trypan blue viability
Trypan blue is excluded by intact membranes, so blue cells are dead. Two caveats: the dye is toxic, so count within 3–5 minutes of mixing, and membrane integrity is a crude proxy — cells well into apoptosis still exclude it. Viability below about 90 % before an experiment usually means something went wrong upstream.
viability (%) = 100 × live / (live + dead)
Frequently asked questions
How many E. coli are in an OD600 of 1.0?
Roughly 8 × 10⁸ cells/mL for a 1 cm path length, though the exact factor depends on strain, growth phase and spectrophotometer geometry — calibrate your own instrument for quantitative work.
How do I calculate cells/mL from a hemocytometer?
cells/mL = (average count per large square) × dilution factor × 10⁴. The 10⁴ comes from the 0.1 mm³ volume above each 1 mm × 1 mm square.
Why is OD600 not linear at high density?
Above about OD 0.8 multiple scattering means light removed from the beam gets scattered back into it, so the reading underestimates true density. Dilute into the linear range and multiply back.