Serial Dilution Calculator
Choose the dilution factor, the number of tubes and the volume in each tube, and get a table of what to transfer, how much diluent to add and the concentration you end up with. A second mode turns colony counts from a plated tube back into CFU/mL of the original sample. For a single dilution, use the Dilution Calculator. Free, no sign-up.
10 means 1 part sample into 9 parts diluent.
1 to 24.
Results
100
Transfer per step (µL)
900
Diluent per tube (µL)
7,200
Total diluent needed (µL)
| Tube | Overall dilution | Concentration | Transfer in | Diluent | Left after next transfer |
|---|---|---|---|---|---|
| 1 | 1:10 (10⁻¹) | 4 × 10⁷ CFU/mL | 100 from stock | 900 | 900 |
| 2 | 1:100 (10⁻²) | 4 × 10⁶ CFU/mL | 100 from previous tube | 900 | 900 |
| 3 | 1:1,000 (10⁻³) | 400,000 CFU/mL | 100 from previous tube | 900 | 900 |
| 4 | 1:10,000 (10⁻⁴) | 40,000 CFU/mL | 100 from previous tube | 900 | 900 |
| 5 | 1:100,000 (10⁻⁵) | 4,000 CFU/mL | 100 from previous tube | 900 | 900 |
| 6 | 1:1,000,000 (10⁻⁶) | 400 CFU/mL | 100 from previous tube | 900 | 900 |
| 7 | 1:10,000,000 (10⁻⁷) | 40 CFU/mL | 100 from previous tube | 900 | 900 |
| 8 | 1:100,000,000 (10⁻⁸) | 4 CFU/mL | 100 from previous tube | 900 | 1,000 |
The working
transfer = volume ÷ factor = 1,000 ÷ 10 = 100
diluent = volume − transfer = 1,000 − 100 = 900
Volumes are in µL. Mix each tube thoroughly before you take the next transfer, and use a fresh tip at every step.
How to Use the Serial Dilution Calculator
- 1. Set the series. Enter the dilution factor per step (10 for a ten-fold series, 2 for a two-fold series) and the number of tubes. Add a starting concentration and its unit if you want the concentration of each tube.
- 2. Set the tube volume. This is the total volume in each tube after mixing. The calculator splits it into the volume to transfer and the volume of diluent.
- 3. Prepare the tubes. Put the diluent volume in every tube, add the transfer volume of stock to tube 1, mix, move the transfer volume from tube 1 to tube 2, and carry on down the row. Copy the table if you want it in your notebook.
- 4. To count bacteria, switch to the CFU mode. Enter your colony counts, pick the tube you plated from and the volume you plated, and read CFU/mL of the original sample.
How a Serial Dilution Works
In a serial dilution each tube is made from the one before it, so the dilutions multiply. With a dilution factor f, each step moves 1/f of the tube's volume into fresh diluent, and the concentration after k steps is the start divided by f to the power k.
Transfer
transfer = tube volume ÷ f
Diluent
diluent = tube volume − transfer
Tube k
concentration = start ÷ fk
Watch the notation. A ten-fold dilution is written 1:10 (or 1/10, or 10−1) as a dilution factor: 1 part sample in 10 parts in total, which is 1 part sample plus 9 parts diluent. The ratio notation 1:9 describes that same mixture. Read a 1:10 dilution as 10 parts of diluent instead and the tube is 11 parts in total, about 9% more dilute at every step, and the error compounds down the series. This calculator always takes the dilution factor, the total divided by the sample part. For a single C₁V₁ = C₂V₂ calculation, use the Dilution Calculator.
Worked Example: A Ten-Fold Series in 1 mL Tubes
You have a culture at about 4 × 10⁸ CFU/mL and want eight ten-fold dilutions in 1,000 µL tubes. Each tube takes 1,000 ÷ 10 = 100 µL of the previous tube and 900 µL of diluent. Tube 1 is 1:10 at 4 × 10⁷ CFU/mL, tube 6 is 1:1,000,000 (10⁻⁶) at 400 CFU/mL, and tube 8 is 1:100,000,000 at 4 CFU/mL. Eight tubes need 8 × 900 = 7,200 µL of diluent in total. These are the calculator's default values.
From Plate Counts to CFU/mL
To count viable cells, spread a known volume of one or more tubes on agar, incubate, and count the colonies. Each colony is assumed to grow from one colony-forming unit (CFU), so the concentration of the original sample is:
CFU/mL
mean colonies × overall dilution factor ÷ volume plated (mL)
With the default counts, 42 and 38 colonies from 0.1 mL (100 µL) of tube 6 (a 10⁶ dilution): the mean is 40, so CFU/mL = 40 × 1,000,000 ÷ 0.1 = 4 × 10⁸. If the sample was diluted before the series — 1 g of food homogenised in 9 mL of buffer, for instance — enter that as the dilution before tube 1 and the answer is per gram.
| Countable range | Source |
|---|---|
| 30–300 colonies | Common textbook convention; AOAC and USDA methods |
| 25–250 colonies | APHA methods |
| 15–300 colonies | ISO 4833; the FDA Bacteriological Analytical Manual since March 2025 |
Too few colonies means a large sampling error, and too many means crowded colonies and missed counts. If you plate several dilutions, use the plate that falls inside the range of the method you are following. The calculator marks each plate against the range you choose. Check which one your lab, course or regulation cites.
Common Mistakes
- Changing the tip too rarely. Carry-over from a concentrated tube inflates every later dilution. Use a fresh tip at each step and mix before transferring.
- Mistaking the ratio for the factor. 1:10 means a total of ten parts, not ten parts of diluent.
- Forgetting the volume plated. Plating 100 µL instead of 1 mL changes the answer ten-fold.
- Averaging plates outside the range. One crowded plate in a set of replicates should not be averaged with the countable ones.
- Ignoring the volume you need to keep. After each transfer a tube holds the tube volume minus the transfer. If you need to plate from the tube afterwards, make the tube volume large enough to cover both.
Frequently Asked Questions
- How do you do a 10-fold serial dilution?
- Put 9 parts of diluent in each tube, add 1 part of the sample to the first tube, mix, then transfer 1 part from tube 1 to tube 2, and so on. In 1 mL tubes that is 900 µL of diluent and 100 µL transferred. Each tube is ten times more dilute than the one before.
- How do you calculate CFU/mL from a serial dilution?
- Divide the average colony count by the volume plated in mL, then multiply by the overall dilution factor of the plated tube. 40 colonies from 0.1 mL of the 10−6 tube gives 40 ÷ 0.1 × 106 = 4 × 108 CFU/mL.
- What is the dilution factor of tube 5 in a 10-fold series?
- The overall dilution factor is 10 raised to the number of steps, so tube 5 is 105, or 1:100,000. Its concentration is the starting concentration divided by 100,000.
- What is the difference between a serial dilution and a single dilution?
- A single dilution goes from stock to the final concentration in one step. A serial dilution reaches very low concentrations in several smaller steps, which is more accurate than pipetting a tiny volume of stock and uses less diluent.
- How many colonies should be on a plate for counting?
- The most common range is 30–300 colonies per plate. APHA methods use 25–250, and ISO 4833 and the FDA Bacteriological Analytical Manual (since March 2025) use 15–300. Choose the range your method specifies.
- How do I make a two-fold serial dilution?
- Set the dilution factor to 2. Each step moves half of the tube volume into an equal volume of diluent, so a 200 µL tube takes 100 µL transferred and 100 µL of diluent. Two-fold series are common for antibody titres, MIC assays and standard curves.