Check remaining cultures

A 1:1 000 dilution worked well for the -8 culture. The remaining cultures from -7 to -4 were seeded with more cells and therefore a dilution factor of 1:10 000 will be used for these overnight cultures. This is achieved through two sequential 1:100 dilutions.

1

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.

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: overnight_-7 , overnight_-6 , overnight_-5 or overnight_-4 ,

After completing the measurement, Access will automatically calculate the bacterial concentration in the cryo stock in the field Cells/mL × Dil.

5

Check remaining cultures

Repeat above steps with 1:10 000 dilutions of the remaining cultures, in this case vial -6, -5, and -4.

Summary

You have now determined the cell concentration in the relevant cultures. Proceed to the next step: Choose best seeding ratio.