Serial Dilution CalculatorTube-by-tube volumes, dilution factors and CFU/mL
Plan a 2-fold, 10-fold or custom dilution series, see how many steps reach your target, and turn plate counts back into CFU/mL.
By Manzoor Ahmad, pharmacist · Updated How do you calculate a serial dilution?
For each tube, transfer volume = final volume ÷ dilution factor, and diluent = final volume − transfer. A 1:10 series with 10 mL per tube moves 1 mL into 9 mL of diluent each step. Tube n then holds C₀ ÷ DFⁿ: from 1 mg/mL, tube 4 is 0.0001 mg/mL, a total dilution of 10⁴.
| Tube | Add from previous | Add diluent | Concentration | Total dilution |
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Serial dilution formulas
Steps to a target: n = log(C₀ ÷ C) ÷ log(DF). If n is not a whole number, use that many full steps and finish with a single partial dilution using the dilution calculator.
How to use the serial dilution calculator
- Plan a series: enter the stock concentration and unit, the dilution factor per step, the number of tubes and the final volume per tube.
- Read the rack: each tube shows what to add and the concentration it ends with.
- Steps to a target: find how many dilutions bring a culture or standard into your working range.
- CFU/mL: enter the colony count, which tube you plated and how much you plated to get the original concentration.
Worked examples
1. Six-tube 10-fold series for a standard curve
From a 1 mg/mL stock, with 10 mL per tube: transfer 1 mL into 9 mL of diluent at each step. Tube 1 is 0.1 mg/mL and tube 6 is 1 × 10⁻⁶ mg/mL (1 ng/mL), a total dilution of 10⁶.
2. Viable count of a bacterial culture
A culture is diluted to 10⁻⁶ and 0.1 mL is spread on a plate, giving 150 colonies. CFU/mL = 150 × 10⁶ ÷ 0.1 = 1.5 × 10⁹ CFU/mL. The count falls in the 30–300 range, so the result is reliable.
3. Two-fold series for an MIC test
With 1 mL per well and a 2-fold factor: transfer 0.5 mL into 0.5 mL of broth each step. Starting at 64 µg/mL, the series runs 32, 16, 8, 4, 2, 1 µg/mL, the classic layout for a minimum inhibitory concentration assay.
Getting accurate serial dilutions
- Mix every tube (vortex or pipette up and down) before taking the next aliquot.
- Change tips each transfer to avoid carry-over, which inflates every downstream tube.
- Keep transfer volumes in your pipette’s best range, typically above 10 µL for a P20 and above 100 µL for a P1000.
- Plate two or three consecutive dilutions so at least one lands in the 30–300 colony window.
Serial dilution FAQs
A serial dilution is a series of step-by-step dilutions where each tube is diluted by the same factor from the one before it. A 10-fold series takes a stock from 1 to 1/10, 1/100, 1/1,000 and so on.
Transfer volume = final volume per tube ÷ dilution factor. For a 1:10 series with 10 mL in each tube, transfer 1 mL into 9 mL of diluent.
Divide the stock concentration by the dilution factor raised to the tube number: Cₙ = C₀ ÷ DFⁿ. Tube 4 of a 1:10 series from 1 mg/mL is 1 ÷ 10⁴ = 0.0001 mg/mL.
The total dilution factor is the product of every step. Three 1:10 steps give 10 × 10 × 10 = 1,000, written 10⁻³ or 1:1,000.
CFU/mL = number of colonies × total dilution factor ÷ volume plated in mL. 150 colonies from the 10⁻⁶ tube with 0.1 mL plated gives 150 × 10⁶ ÷ 0.1 = 1.5 × 10⁹ CFU/mL.
Count plates with about 30 to 300 colonies. Fewer than 30 is statistically unreliable, and more than 300 is usually too crowded to count accurately (TNTC, too numerous to count).
They usually mean the same thing in lab practice: 1 part sample in 10 parts total. Some protocols use 1:9 to mean 1 part sample plus 9 parts diluent, which is also a 10-fold dilution.
Large single dilutions need tiny, inaccurate volumes or huge amounts of diluent. Several smaller steps keep every pipetting volume in an accurate range.
Mix equal volumes of sample and diluent at each step. With 1 mL final volume, transfer 0.5 mL into 0.5 mL of diluent, then repeat.
Steps = log(C₀ ÷ C_target) ÷ log(DF). Going from 10⁸ to 10² cells/mL in 10-fold steps takes log(10⁶) ÷ log(10) = 6 steps.
Yes. Use a fresh tip for every transfer and mix each tube thoroughly before taking the next aliquot. Carry-over from a used tip is one of the most common sources of error.
When you transfer liquid out of a tube to make the next one, that tube is left with less. The option increases the transfer and diluent volumes so every tube keeps the same final volume after its transfer.
Sources
- American Society for Microbiology: Serial Dilution Protocols
- U.S. FDA Bacteriological Analytical Manual, Chapter 3: Aerobic Plate Count
- OpenStax Microbiology, Section 9.1: How Microbes Grow (viable plate count)
- Sanders ER. Aseptic Laboratory Techniques: Plating Methods. J Vis Exp. 2012
- NIST SP 811: Guide for the Use of the International System of Units
This serial dilution calculator is for education and lab planning. Follow your laboratory’s validated SOPs for clinical, food safety or regulated testing.