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 in 99ml diluent)

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

2. Pipet 0.9ml of sterile water into each tube.

3. Transfer 0.1 ml of the 1:100 diluted Soil Extract to your tube labeled 10^-3 , and mix well by vortexing or shaking vigorously.

4. Transfer 0.1 ml of the 10^-3 dilution to the next tube with 0.9ml 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. Label (as appropriate: single,duplicate or triplicate) dilute nutrient agar plates 10^-5, 10^-6, and 10^-7.

7. For Culturable colony counts of the dilutions: 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.