Inoculate culture

Once you know the cell concentration of the inoculum you can calculate the volume necessary to hit a target seeding concentration (here 1 × 10⁷ cells/mL). Use the subcultivation calculator tool for the calculations.

Use Optiseeding workflow to determine suitable seeding concentration

The seeding concentration may need adjustment to get a high-quality culture. Do a screening experiment with a broad 1:10 dilution series to find a suitable seeding concentration. See Optimize starter culture for more details.

1

Open the subcultivation calculator

Open the subcultivation calculator tool in a browser.

2

Calculate inoculation volume

• Fill in cells/mL of the inoculum in the first field.
• Add volume information in the second field. In this case it is 50 mL
• Set the desired inoculation concentration in the final field. In this case 1 × 10⁷ cells/mL
• Click Calculate V_add to get the result.

3

Add inoculum culture to growth medium

Transfer the calculated volume of inoculum to growth curve flask and swirl aggressively to disperse the inoculum.

Record the inoculation time. Note it down on the autoclave tape as a backup, ensuring you have a reference if you forget to document it elsewhere.

Inoculation of shake flask at well-defined concentration. In this video the 50 mL vial with the red cap represents the inoculum.

Summary

Once the growth medium is inoculated, proceed to Determine initial concentration.