Enter the number of colonies, dilution factor (the reciprocal of the dilution), and volume plated into the calculator to determine the bacterial concentration.
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Bacterial Concentration Formula
The calculator uses one of three formulas depending on the tab selected.
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
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
cells/mL = OD600 × F × Df
- OD600 = optical density reading at 600 nm
- F = conversion factor (cells/mL per OD unit)
- Df = sample dilution factor before reading (1 if undiluted)
Plate-count math assumes each colony came from one viable cell, so results are CFU/mL rather than total cells/mL. The OD600 formula assumes a linear range, typically OD 0.1 to 0.8; above that, dilute and re-read. Conversion factors are species- and instrument-specific, so calibrate against your own plate counts when accuracy matters.
Reference Tables
Typical OD600-to-cell-density factors for common lab organisms:
| Organism | Cells/mL per OD600 |
|---|---|
| E. coli | 8 × 10^8 |
| Salmonella | 1 × 10^9 |
| Bacillus subtilis | 7 × 10^8 |
| Pseudomonas aeruginosa | 1 × 10^9 |
| S. cerevisiae (yeast) | 3 × 10^7 |
| General estimate | 1 × 10^9 |
How to read your colony count:
| Colonies on plate | Interpretation |
|---|---|
| < 30 | Estimate only; plate a lower dilution |
| 30 – 300 | Statistically valid count |
| > 300 | Crowded; plate a higher dilution |
| TNTC | Too 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 doesn’t OD600 match my plate count? OD measures all cells that scatter light, including dead and non-culturable cells. Plate counts only capture viable, dividing cells. A 2-fold to 10-fold gap between the two is common.
Should I count colonies on more than one plate? Yes. Plate in duplicate or triplicate at the dilutions likely to fall in the 30 to 300 range, then enter the average colony count.
What if my OD reads above 1.0? The relationship between OD and cell density is no longer linear. Dilute the sample 1:10 in fresh media or saline, re-read, and enter 10 in the sample dilution factor field.
