What a serial dilution is
A serial dilution is a chain of dilutions where each tube is made from the one before it. It reaches very large dilutions, such as one in a million, with volumes that are easy to pipette, and it gives a set of evenly spaced concentrations for standard curves and plate counts.
How to do a 1:10 serial dilution
- Label the tubes and add 900 µL of diluent (water, buffer or medium) to each.
- Add 100 µL of the sample to the first tube and mix well by pipetting or vortexing. This tube is a 1:10 (10−1) dilution.
- With a fresh tip, move 100 µL from the first tube to the second and mix. This is 10−2.
- Repeat down the row. Each tube is ten times weaker than the one before.
For a twofold series, used for standard curves and antibody titrations, mix equal volumes instead, such as 500 µL of the previous tube into 500 µL of diluent.
Working out the total dilution
The dilution factor of one step is total volume ÷ volume transferred: (100 + 900) ÷ 100 = 10. The total dilution of a tube is the step factors multiplied together, so the sixth tube of a 1:10 series is diluted 106 times. Its concentration is the starting concentration ÷ 106.
Counting colonies from a dilution
Plate a known volume from a few tubes and count the plate with 30 to 300 colonies. Then:
CFU per mL = colonies × total dilution ÷ volume plated (mL)
45 colonies from 0.1 mL of the 10−6 tube give 45 × 106 ÷ 0.1 = 4.5 × 108 CFU/mL in the original sample.
Tips for accurate dilutions
- Change tips between tubes; carried-over liquid adds error that grows down the series.
- Mix each tube thoroughly before taking the next transfer.
- Pipette volumes in the middle of the pipette's range, not at its smallest setting.
Calculators
The Serial Dilution Calculator lists the concentration of every tube and how much to transfer and add. For a single dilution, use the Dilution Calculator; to count cells before diluting, use the Hemocytometer Calculator.