BISC209: Serial Dilution

Serial Dilution Technique :

This technique is useful for obtaining countable numbers of bacterial colonies by diluting a highly concentrated culture. The protocol described here is for serial 1:10 dilutions and can be modified for any dilution series desired. Generally, the last 3 dilutions are plated in duplicate or triplicate. This procedure is based on using a 1/100 stock (e.g. Soil Extract of 1 gram with diluent added to 100 ml)

1. Label 5 tubes 10^-3, 10^-4 etc. through 10 ^-7.

2. Label destination plates of appropriate solid medium with dilution and identifying information. Because your goal is to obtain 30-300 well isolated colonies on a plate, generally only the 10^-5 through 10^-8 dilutions are plated. If your soil is exceptionally rich or poor the choice of dilutions will need to be modified. Often dilutions are plated in duplicate or triplicate.

2. Pipet 0.9 ml of sterile water using the same sterile 1 ml pipet into the 5 tubes labeled in step 1.

3. Using a new tip, transfer 100 μL of the 1:100 diluted Soil Extract to your tube labeled 10^-3 using your p200 micropipette, and mix well by vortexing or shaking vigorously.

4. Transfer 100 μL ml of the 10^-3 dilution to the next tube containing 0.9 ml sterile water to make a 10,^-4 , and mix well.

5. Continue sequentially in this manner until you have carried the dilution to 10^-7.

6. 7. Use a flamed glass spreader, spread a 1ml (or 0.1ml) sample of the 10^-5 dilution on the agar surface of the appropriately labeled dish.

8. Repeat for 10^-6 and 10^-7dilution.

9. Allow any moisture to be absorbed into the agar, invert and incubate, at the appropriate temperature.