About the Serial Dilution Calculator
This serial dilution calculator plans a stepwise dilution series. Enter the stock concentration, the dilution factor for each step, the number of steps and the volume you want in each tube, and it tells you how much to transfer and how much diluent to add, plus the concentration and cumulative dilution of every tube. If you already know the final concentration you need, switch to target mode and it works out the per-step dilution factor for you.
It is used for standard curves in ELISA and qPCR, bacterial plate counts (CFU/mL), minimum inhibitory concentration (MIC) assays with two-fold series, and any time a single dilution would require pipetting an impractically small volume. Concentration units are just labels — the maths is identical for molarity, mass per volume, cells or copies.
The calculator assumes each tube is made by adding the transfer volume from the previous tube to fresh diluent so that each tube holds the chosen total volume before the next transfer. Each intermediate tube therefore ends up with the total volume minus one transfer, while the last tube keeps the full volume.
How to use the serial dilution calculator
- 1Enter the stock concentration and choose a unit label.
- 2Choose a dilution factor per step, or switch to target mode and enter the final concentration you need.
- 3Set the number of steps and the volume you want in each tube.
- 4Fill each tube with the diluent volume shown, then transfer the given volume tube to tube, mixing each time.
- 5Use the table to label tubes with their concentration and cumulative dilution.
Formula and method
In a serial dilution each tube is diluted by the same factor DF relative to the previous one, so after n steps the concentration has fallen by DFⁿ. A ten-fold series of five steps dilutes a sample 100,000 times. To make each tube, a volume V_total ÷ DF is carried over from the previous tube and topped up with diluent to V_total, which is C₁V₁ = C₂V₂ applied at every step.
In target mode the per-step factor is the n-th root of the overall dilution needed. Because errors compound from tube to tube, thorough mixing and accurate pipetting at every step matter; using a fresh tip for each transfer avoids carry-over.
- C₀
- Stock concentration
- Cₙ
- Concentration after n dilution steps
- DF
- Dilution factor per step (10 for a 1:10 dilution)
- n
- Number of dilution steps
- V_total
- Volume in each tube after mixing, before the next transfer
Worked examples
Five-step ten-fold series from 100 µg/mL
Each step is 1:10, so 100 µL is carried into 900 µL of diluent. After five steps the total dilution is 10⁵ and the last tube holds 100 ÷ 100,000 = 0.001 µg/mL.
Two-fold MIC series to reach 2 µg/mL
Going from 64 to 2 µg/mL is a 32-fold dilution; spread over five steps that is 32^(1/5) = 2 per step. Each well receives 100 µL diluent and 100 µL from the previous well, giving 32, 16, 8, 4 and 2 µg/mL.
Bacterial culture for plate counts
A culture at about 2.5 × 10⁹ CFU/mL diluted 1:10 seven times (1 mL into 9 mL each step) reaches 250 CFU/mL. Plating 0.1 mL of the last tube would give roughly 25 colonies, inside the countable range.
Frequently asked questions
How do you calculate a serial dilution?+
Divide the starting concentration by the dilution factor once for every step: Cₙ = C₀ ÷ DFⁿ. Three ten-fold dilutions of a 1 M solution give 1 ÷ 10³ = 0.001 M (1 mM).
What does a 1:10 dilution mean?+
One part sample in ten parts total — for example 100 µL of sample plus 900 µL of diluent. Some older texts use 1:10 to mean one part sample to ten parts diluent (an 11-fold dilution), so check the convention used in your protocol.
Why use a serial dilution instead of one big dilution?+
A million-fold dilution in one step would need 1 µL of sample in a litre of diluent, which is wasteful and imprecise. Several smaller steps keep volumes practical and give you every intermediate concentration for a standard curve or plate count.
How do I calculate the original concentration from a plate count?+
Multiply the colony count by the total dilution factor and divide by the volume plated. 42 colonies from 0.1 mL of a 10⁻⁶ dilution means 42 × 10⁶ ÷ 0.1 = 4.2 × 10⁸ CFU/mL in the original sample.
How accurate is a serial dilution?+
Pipetting errors multiply through the series, so a 2% error per step becomes about 10% after five steps. Mix each tube thoroughly, use calibrated pipettes and change tips between transfers.