SERIAL DILUTION

A serial dilution lowers the concentration of a sample in equal steps, for example 1:10 each time. It is used when a culture is too dense to count or plate directly, so that at least one dilution gives a countable number of separate colonies.

WHAT YOU NEED?

• Sample (culture or library)

• Sterile diluent (LB broth, saline or water)

• Sterile tubes and a micropipette with sterile tips

• Marker

STEPS

1. Label tubes with each dilution (1:10, 1:100, 1:1000 and so on).

2. Add the diluent to each tube; (for 1:10 steps, 900 µL per tube).

3. Add (100 µL) of the sample to the first tube and mix well. (This is a 1:10 dilution).

4. With a fresh tip, move 100 µL from the first tube into the second and mix. This is 1:100.

5. Repeat down the series, changing tips every time.

6. Plate a set volume from the dilutions you want to test.

7. After incubation, count plates with roughly 30–300 colonies and multiply by the dilution factor to estimate the original concentration.

TIPS

• Mix each tube thoroughly before taking the next transfer.

• Changing tips each time is essential; carry-over throws off every later dilution.

• A 1:2 dilution works the same way, using equal volumes of sample and diluent.