Before you start
- Label tubes 10⁻¹ to 10⁻⁶ and plates with sample and dilution.
Reagents and materials
Totals are for 3 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| Sterile diluent (PBS or saline) for six 10-fold dilutions900 µL per tube | 5.4 mL | 5.4 mL |
| Agar plates | 4 each | 4 each |
| Sample | 100 µL | 100 µL |
Buffers and recipes
Calculation per sample
| Countable plates | 30–300 colonies (spread plates) |
| CFU/mL | colonies ÷ (dilution × volume plated in mL) |
| Example | 150 colonies from 100 µL of the 10⁻⁵ dilution → 150 ÷ (10⁻⁵ × 0.1) = 1.5 × 10⁸ CFU/mL |
Equipment
- Sterile tubes
- Spreader or glass beads
- Incubator
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Prepare tubes
Add 900 µL diluent to each tube.
Dilute
Transfer 100 µL sample into the first tube, vortex, change tip, and repeat down the series.
Plate
Spread 100 µL of the appropriate dilutions on plates (duplicate), or spot 10 µL drops for a quick estimate.
Incubate
Incubate inverted at the right temperature until colonies are countable (often 16–24 h).
Count
Count plates with 30–300 colonies and calculate CFU/mL.
Critical points
- Change tips at each dilution and mix well — the most common sources of error.
Troubleshooting
| Problem | What to check or change |
|---|---|
| All plates too many or too few | Adjust the dilution range. |
| Spreading colonies | Use drier plates; plate smaller volumes. |
Safety
Work at the organism’s biosafety level; decontaminate plates before disposal.
Further reading
- ASM Microbe Library: serial dilution and plate count protocols.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.