Morning measurement

Incubate the cultures until they are all at maximal cell concentrations. For E. coli an incubation time of 24 hours is sufficient. We expect the concentration to be between 1 × 10¹⁰ and 5 × 10¹⁰ cells/mL and therefore a 1:10 000 dilution is usually suitable to get within the BactoBox® upper working range of 5 × 10⁶ total/mL.

The below video demonstrates the sample workup.

1

Retrieve a sample

Use a serological pipette to sample 1 mL culture. Transfer to a 5 mL centrifuge tube. Note down the sampling time.

2

Disaggregate cell clumps

Vortex the vial 1 minute at maximum speed to disaggregate cell clumps. See Break up clumps and chains if your bacterium needs more aggressive disaggregation than this.

3

Dilute 1:100

Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.

4

Dilute 1:10 000

Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.

5

Measure the diluted sample on BactoBox®

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

Press Measure in Access to start a new measurement.

• Dilution: 10 000

• Label: Growth_medium_type . For example LB or TSB.

After completing the measurement, Access will automatically calculate the cell concentration in the culture in the field Cells/mL × Dil.

6

Check remaining cultures

Repeat above steps with 1:10 000 dilutions of the remaining growth media.

Pro tip: Add information on incubation time to each growth medium type (label)

The Access column calcHPI (calculated hours post inoculation) is a straightforward way to append incubation time to your measurements. If your cultures were inoculated 24 hours before the first measurement, then add the number 24 to the HPI cells for each label. This will automatically offset the measurements with 24 hours. This is optional, but very convenient.

Summary

You have now determined the cell concentration for the morning measurement. Wait ~4 hours before proceeding to Noon measurement.