Measure on cryo stock

The cryo stock is the starting point for preparing the dilution series. It will likely contain a mix of culturable, viable-but-non-culturable (VBNC), and dead cells. Knowing the cell concentration helps determine how far the dilution can go before cells are virtually absent in the growth medium. We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.

1

Disaggregate cell clumps

Allow the cryo stock to thaw at room temperature.

Vortex the cryo stock 1 minute at maximum speed to disaggregate cell clumps.

2

Dilute 1:100

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

3

Dilute 1:1000

Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.

Transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent. Vortex 10 seconds at maximum speed.

4

Measure on the diluted sample

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

PressMeasure in Access to start a new measurement.

• Dilution: 1 000

• Label: E_coli_cryo

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

Summary

Now you know the bacterial concentration of your cryo stock. Next step: Prepare dilution series.