Same-day subculture (strategy 1)

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 as a seed.

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.