Calculate CFU/mL from colonies, dilution, and plated volume, or plan expected colonies and target dilutions with clear fraction and factor inputs.

CFU is a colony-forming-unit estimate, not a total cell count. Dilution is entered as either sample fraction or its reciprocal. Countability and OD600 calibration depend on the validated method.

Use zero for no observed colonies. A replicate mean may be fractional.

For 10⁻⁵, enter 0.00001 as a fraction or 100000 as a reciprocal factor.

Bacterial Concentration Formula

The four solve targets calculate original CFU concentration, expected plate colonies, a target dilution fraction, or an empirical OD600-based CFU estimate. A sample fraction of 0.00001 and a reciprocal factor of 100000 both represent a 10⁻⁵ dilution.

Plate count

CFU / mL = N / (D × V)
  • N = colonies counted on the plate
  • D = dilution plated (e.g. 10^-6 = 0.000001)
  • V = volume plated, in mL

Serial dilution: derive the overall fraction first

For equal serial steps, the overall fraction is (transfer volume / final mixed volume) raised to the number of steps. Enter that final fraction or its reciprocal in the plate-count calculator; it has no separate serial-step controls.

CFU / mL = N / ((Vt / Vf)ⁿ × Vp)
  • N = colonies counted
  • Vt = transfer volume per step
  • Vf = final tube volume after mixing
  • n = number of serial dilution steps before plating
  • Vp = volume plated, in mL

OD600 estimate

Estimated CFU/mL = (sample OD600 − blank OD600) × empirical calibration.

  • OD600 = optical density reading at 600 nm
  • Calibration = validated CFU/mL per corrected OD600 for the same organism and measurement conditions
  • This OD mode reports the sample represented by the calibration. It has no separate pre-reading sample-dilution-factor field.

A CFU can originate from a single cell or an aggregate capable of forming a colony under the selected conditions. It is not a total cell count. OD600 measures light scattering and may include nonviable cells or debris. Its usable range and conversion to CFU require empirical calibration for the organism, medium, growth state, instrument and optical path; a cells/mL factor is not automatically a CFU/mL factor.

Reference Tables

There is no universal OD600-to-CFU conversion factor. Use a validated calibration rather than a generic species value. See Stevenson et al. (2016) and Beal et al. (2020).

The 30–300 range below is a common illustrative convention, not a universal validity rule. The FDA BAM Chapter 3 (January 2026) uses 15–300 for its method. Follow the limits and reporting rules of the method used:

Colonies on plate Interpretation
< 30Estimate only; plate a lower dilution
30 – 300Within this illustrative convention; follow the validated method
> 300Crowded; plate a higher dilution
TNTCToo numerous to count; rerun with more dilution

Worked Example

You plated 100 µL of a 10^-6 dilution and counted 125 colonies.

CFU/mL = 125 / (10^-6 × 0.1 mL) = 125 / 10^-7 = 1.25 × 10^9 CFU/mL

Why does OD600 differ from my plate count? The two methods measure different properties. OD measures scattering; plate counts measure colony-forming units under the selected conditions. There is no universal ratio between them.

Can I use a replicate mean? A simple average is appropriate here only when replicates have the same dilution and plated volume. Results from different dilutions or volumes require the pooling or weighting rule specified by the validated method.

What if the OD reading is outside the calibrated range? Follow the validated measurement method. This mode does not provide a universal linearity threshold or a field for a separate measurement dilution; account for any known pre-reading dilution when interpreting the measured-sample result.