BactoBox measurements

Once the cell clumps are disaggregated and the sample is cold, perform the BactoBox® measurements in triplicate.

We expect a high cell concentration and therefore an 1:10 000 dilution is usually sufficient. Dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a 1:10 000 dilution.

The video below demonstrates the sample preparation.

Step by step

1

Vortex briefly

Vortex the sampling vial 5 seconds at max speed to ensure that cells are evenly dispersed. This assumes that the sample was already aggressively disaggregated in the previous step Retrieve culture sample.

2

Dilute 1:100

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

3

Dilute 1:10 000

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

4

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: End-of-fermentation .

• Sampling time: Register the exact time when the sample was obtained, i.e. what you wrote on the sampling vial.

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

5

Measure repeat #2 and #3

Repeat steps 1–4 two additional times to obtain triplicate repeats.

Use exactly the same input information as in step 4. When identical sampling time and label is used, Access will treat the data as replicates.

Pro tip: Add information on incubation time .

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 BactoBox® cell concentration in triplicate repeats for the EoF sample. Next step is Calculate dilutions.