Low-inoculum overnight culture (strategy 2)

Use this workflow when you want a low-inoculum overnight culture that reaches late exponential or early stationary phase by morning so BactoBox counts match CFU at the time you dose.

Concept

You inoculate the overnight culture with a very low cell concentration. This stretches the growth curve so that after about 16 hours the culture is in late exponential or early stationary phase, not in deep death phase. In that phase, BactoBox cells/mL aligns well with CFU.

When to use

Use this strategy when:

• You want a culture that is ready at a fixed time next day (for example after ~ 16 hours).

• You know the approximate doubling time from the literature or prior runs.

• You prefer to set and forget the overnight culture once you have validated it.

Choosing the inoculum concentration

Run the Get high-quality overnight cultures from cryo stocks protocol to identify the optimal inoculum concentration for your overnight culture. If you want a quick starting point, use the table below as a rule of thumb for a 16 hour overnight culture in rich media.

Doubling time (approx.)	Target inoculum in overnight culture
≤ 30 minutes	10 cells/mL
~ 40 minutes	1 × 10³ cells/mL
~ 60 minutes	1 × 10⁴ cells/mL
~ 80 minutes	1 × 10⁵ cells/mL

If you extend the overnight incubation (for example to 24 hours), you should reduce the inoculum further to avoid deep death phase and then re validate the protocol.

Step-by-step

1. Prepare a seed suspension

   • If you inoculate from a cryostock and you know the approximate CFU/mL for that batch, you can base your calculation directly on that.

   • If you inoculate from a plate, prepare a seed suspension (for example by resuspending colonies in medium or buffer) and measure it with BactoBox.

2. Measure the seed suspension with BactoBox

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

   • Measure and record cells/mL for the seed suspension.

3. Set the inoculum concentration for the overnight culture

   • Choose the target inoculum cells/mL from the table above based on your best estimate of doubling time.

   • Calculate the volume of seed suspension you need to add, $V_{add}$, to the overnight culture volume:

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

where:

• $C_{target}$ is the target inoculum concentration in the overnight culture in cells/mL (for example 10 cells/mL).

• $V_{overnight}$ is the volume of media in the overnight flask or bottle (mL) before adding seed suspension.

• $C_{seed}$ is the cells/mL of the seed suspension as measured by BactoBox®.

Alternatively, use our online calculator:

BactoBox® Low-inoculum Overnight Calculatorsbtinstruments.com

Often the calculated volume is too small to pipette accurately. In these cases, prepare an intermediate dilution of the seed suspension instead of attempting to transfer very small volumes.

Example: If $V_{add}$ is 0.2 µL, dilute the seed suspension 1:100 and then add 20 µL instead.

1. Inoculate and incubate

   • Add the calculated volume of seed suspension to the overnight medium.

   • Incubate with shaking for 16 hours (or the time that you plan to use and validate).

2. Measure the overnight culture just before dosing

   • Take a well‑mixed sample from the overnight culture at the planned harvest time.

   • Chill the sample on ice to halt growth if you will also plate it (see validation tips on the High-accuracy microbial input page).

   • Prepare dilutions and measure cells/mL with BactoBox.

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