dilutioncalculator.co.uk§ Verified 20 Jun 2026 · Reviewed by A. Hargreaves, MChem (Hons)
Serial dilutions, step by step
A 10-fold serial brings a thick microbial broth down to a plate-countable concentration in six 1 ml transfers. The method is robust because each step is in a forgiving pipetting range.
§ 1Set-up
- Six labelled 10 ml tubes, each with 9 ml of sterile diluent (saline or peptone water).
- One sample tube. P1000 pipette with sterile tips. Vortex.
- Sterile loop or spread plate at the end.
§ 2Procedure
- Vortex the sample. Transfer 1 ml into tube 1 (1 ml + 9 ml = 10⁻¹).
- Vortex tube 1, fresh tip. Transfer 1 ml into tube 2 (10⁻²).
- Repeat tubes 3 through 6, giving 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶.
- Plate 100 µl from each of the last three tubes onto agar.
- Incubate. Count plates with 30 to 300 colonies. Back-calculate.
§ 3Back-calculation
If tube 5 (10⁻⁵) plated 100 µl yields 122 colonies, the working concentration in that tube was 122 ÷ 0.1 ml = 1 220 cfu/ml. Original concentration = 1 220 × 10⁵ = 1.22 × 10⁸ cfu/ml.[22]
§ 4Common errors
- Re-using a tip across steps. Even invisible carry-over distorts the count by an order of magnitude at the dilute end.
- Inadequate mixing. Pipette-mix five times after each transfer, or vortex 5 seconds.
- Plating before the agar surface dries. Spread evenly, leave 10 minutes upright.