Use 1:10 000 dilutions at high concentrations

Use a 1:10,000 dilution if the 1:1,000 dilution results in more than 1,500,000 total/ml, as observed in the sample analyzed approximately 30 minutes ago.

Do a Clean program after each measurement.

Start a 30-minute countdown to remind you to collect the next sample.

1

Prepare a 1:10 000 dilution

1. Use a serological pipette to transfer a sample from the shake flask to a vial.

2. Use a permanent marker to label the vial cap with the sampling time.

3. Return the shake flask to the incubator shaker.

4. Start a 30-minute countdown to remind you to collect the next sample.

5. Vortex 30 seconds at max speed to disaggregate bacteria.

6. Prepare a 1:10 000 dilution using two sequential 1:100 dilution steps.

   1. First make a 1:100 dilution:

      1. Transfer 101 µL of your sample to 10 mL of diluent.

      2. Vortex 10 seconds at maximum speed.

   2. Then make the 1:10 000 dilution:

      1. Transfer 101 µL of the 1:100 dilution to 10 mL diluent.

      2. Vortex 10 seconds at maximum speed.

7. Transfer the tubing kit to the 1:10 000 vial.

2

Measure the diluted samples in Access

1. Click the Measure button in Access.

2. Add metadata:

   1. Dilution: 10 000.

   2. Label: E_coli_growth.

3

Clean the device

1. Transfer the tubing kit to the disinfection vial.

2. Click Clean.

Summary

You have now tracked the concentration through lag, exponential, deceleration and stationary stage. The final step is to determine when to conclude the growth curve experiment.