Same-day subculture (strategy 2)

Use this workflow when you have an overnight culture in the morning and need a refreshed, exponential-phase culture whose BactoBox counts match CFU by midday.

Concept

You take an existing overnight culture, use BactoBox to set the inoculum for a subculture in fresh medium, and then grow this subculture for a few hours. This gives you a same day culture that is dominated by active, culturable cells.

When to use

Use this strategy when:

• You have an overnight culture available in the morning.

• You want a refreshed culture that is in exponential growth around midday.

• The organism grows fast (typical doubling time ≤ 40 minutes in your medium).

Step-by-step

1. Prepare the overnight culture Prepare an overnight culture using your usual protocol. In this workflow you only use it to inoculate your subculture.

2. Measure the overnight culture with BactoBox in the morning

   • Prepare an appropriate dilution series (for example 1:100, 1:1,000, 1:10,000) so that total/mL lands below 5,000,000 in at least one dilution.

   • Measure and record dilution-adjusted cells/mL for the culture.

3. Calculate the inoculum volume Calculate how much volume to add, $V_{add}$, to your fresh shake flask to reach 1e6 cells/mL starting concentration:

$V_{\text{add}} = \frac{C_{\text{target}} \times V_{\text{subculture}}}{C_{\text{overnight}}}$

where:

• $C_{target}$ is the target concentration in the subculture, 1e6 cells/mL as default

• $V_{subculture}$ is the initial volume (mL) of fresh media in the new shake flask

• $C_{overnight}$ is the diluted-adjusted concentration of cells/mL in the overnight culture as measured by BactoBox®. Alternatively, use our online sub-cultivation calculator.

BactoBox® Sub-Cultivation Calculatorsbtinstruments.com

1. Inoculate and grow the subculture

   • Add the calculated inoculum volume to the fresh medium.

   • Incubate with shaking for about 3–4 hours.

   • For very fast growers, lean towards 3 hours; for slower ones, lean towards 4 hours or slightly more.

2. Measure the subculture just before dosing

   • Take a well‑mixed sample from the subculture.

   • Prepare dilutions so that total/mL is within range.

   • Measure cells/mL with BactoBox.

   • Adjust the result for the dilution factor.

3. Calculate the dosing volume Use the approach outlined on the High-accuracy microbial input page to calculate the dosing volume.