Determine starter culture concentration

You need a starter culture with a known concentration. From this, we calculate the volume required to reach a final concentration of 1 × 10⁷ cells/mL in the flask.

Here we have already prepared the overnight culture based on the guidance provided in Get high-quality overnight cultures from cryo stocks.

Retrieve a sample of the starter culture

1. Use a serological pipette to aseptically transfer 10 mL of the overnight culture to a sterile vial.

2. Vortex thoroughly to disperse any cell clumps. For an E. coli culture it is usually sufficient to vortex 30 seconds at max speed.

Proper vortexing is important for reliable results!

Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.

Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.

Prepare the sample for BactoBox® measurement

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

   1. 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. Make a 1:10 000 dilution:

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

      2. Vortex 10 seconds at maximum speed.

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

Measure the sample in Access

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

2. Click Measure to start a new measurement. Notice that there is a group icon on the button because you are within a measurement group.

   1. Dilution: 10 000

   2. Label: Overnight_culture

3. After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.

Clean the device

1. Transfer the tubing kit to the disinfection vial.

2. Click Clean. The button in Access works the same way as the one on BactoBox®.

Summary

Now you know the bacterial concentration of your starter culture. Proceed to the next step to determine the particle concentration in the non-inoculated growth medium.