Determine concentration after incubation

For every e.g. 2 hours, pull a sample to check if the cell concentration has increased. The starter culture is ready when the cell concentration is at least ten-fold higher.

Extend incubation until cell concentration is at least ten-fold higher compared to the T₀ result

• A cryo stock contains a complex mix of culturable, viable-but-non-culturable, and dead cells. The initial BactoBox® cell concentration right after inoculation detects all these variants and does therefore not only reflect the concentration of culturable cells.

• Incubate the culture until the cell concentration is at least a 10× higher. At this point at least 90% of the detected cells are culturable.

Step by step

1

Retrieve a sample after 2 hours

After 2 hours of incubation, use a serological pipette to pull 1 mL sample. Transfer to e.g. a 5 mL centrifuge vial.

2

Disaggregate cell clumps

Vortex the sample 1 min at max speed to disaggregate cell clumps.

3

Dilute 1:100

Transfer 101 µL of T0 culture to 10 mL of diluent. Vortex 10 sec. at max speed.

4

Measure

Transfer tubing kit to the diluted sample. Press Measure and wait for the result to appear on the BactoBox® screen.

5

Calculate concentration

Multiply the cells/mL result with the dilution factor, in this case 100, to get the cell concentration in the culture at T₀.

6

Stop or repeat

Your starter culture is ready when the cell concentration is at least ten-fold higher than at T₀,

If the concentration is still too low, extend the incubation by two hours and repeat steps 1 to 5 until ten-fold increase is obtained.

Summary

The starter culture is ready once the cell concentration has increased by at least 10-fold. You can now use the culture for experiments or to continue with the next step in your seed train.