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OD600 to Cell Density Calculator

Correct an OD600 reading for dilution and convert it to an estimated cell density using a conversion factor you can set for your own strain and instrument.

Formula

OD600culture=OD600read×DF\mathrm{OD}_{600}^{\text{culture}} = \mathrm{OD}_{600}^{\text{read}} \times \mathrm{DF}
cells/mL≈OD600culture×F\text{cells/mL} \approx \mathrm{OD}_{600}^{\text{culture}} \times F
OD600read\mathrm{OD}_{600}^{\text{read}}
OD600 of the diluted sample, blank subtracted
FF
cells per mL at an OD600 of 1, specific to the organism and instrument

How it works

Optical density at 600 nm measures light scattered by cells, not light absorbed, so it depends on cell size and shape and on the geometry of the instrument. Within the linear range it rises in proportion to cell density.

The conversion to cells per mL is empirical. 8 × 10⁸ cells/mL per OD unit is a figure often quoted for E. coli, but the true value varies with strain, growth phase, medium and spectrophotometer. Calibrate against plate counts or a counting chamber for anything quantitative.

Worked example

A culture diluted 1:10 reads 0.32, using 8 × 10⁸ cells/mL per OD unit.

  1. Culture OD600 = 0.32 × 10 = 3.2.
  2. cells/mL ≈ 3.2 × 8 × 10⁸ = 2.56 × 10⁹.

The culture is at OD600 3.2, roughly 2.56 × 10⁹ cells/mL.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The reading is within the linear range of the instrument, which is why dense cultures are diluted first.
  • The conversion factor matches the organism, growth phase and instrument.
  • The blank is the same medium as the culture.

Common mistakes

  • Reading a dense culture undiluted. Above the linear range, OD underestimates cell density.
  • Reusing a conversion factor from another instrument. Path length and optics differ, especially between cuvettes and microplates.
  • Treating OD as a viable count. Dead cells scatter light too.

Related tools

Bacterial Growth Rate CalculatorSpecific growth rate and doubling timeOpen calculatorCFU Calculator (Colony-Forming Units per mL)Viable count from a plate count and dilutionOpen calculatorCalibration Curve Calculator (Standard Curve)Linear fit, residuals and an unknown with its uncertaintyOpen calculatorSerial Dilution CalculatorTransfer volumes and the concentration in every tubeOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.