# Overnight cultures

How you seed an overnight culture affects culture quality. Your culture may stay [high-quality](/mpd/encyclopedia/high-quality-culture.md), or it may become dominated by stressed, non-culturable cells.

{% hint style="success" %}
A [high-quality culture](/mpd/encyclopedia/high-quality-culture.md) has a high proportion of culturable bacterial cells.
{% endhint %}

{% hint style="danger" %}
A low-quality culture has a low proportion of culturable bacterial cells.
{% endhint %}

## Introduction

It is common lab practice to use ≥1 % (v/v) inoculum volume. This works for same-day expansion. This can ruin overnight cultures.

Fast-growing species can finish exponential growth in about 6 h. After that, the culture enters stationary and death phases. The culture is often low-quality the next morning.

Prevent overseeding by starting lower. This prolongs exponential growth before nutrient depletion.

## Method

We did a simple experiment with stationary-stage *E. coli* ATCC 8739.

We seeded 10 shake flasks with different inoculum volumes:

* 1 % v/v (10<sup>-2</sup>) at the high end.
* 0.00000001 % v/v (10<sup>-11</sup>) at the low end.

We achieved the lowest inoculum via a dilution series across flasks. **Key step:** transfer **5 mL** from the preceding flask into **45 mL** fresh medium in a **new** flask.

We incubated at 37 °C and 150 rpm. We analyzed cultures at 10 h, 18 h, and 24 h.

We assessed culture quality by comparing culturable cells (CFU/mL) with total cell concentration (BactoBox® BacTotal-2024-10 with an ECC factor of 1.27). A ratio close to 1 (100 %) indicates a [high-quality culture](/mpd/encyclopedia/high-quality-culture.md). To account for CFU variability, we considered 80–125 % as high-quality.

## Results

The experiment showed clear differences in culture quality depending on inoculum size:

* **At 10 h post inoculation**, all cultures were healthy. Culturable cell counts and total cell counts matched closely.
* **At 18 h post inoculation**, cultures seeded at 1 % (10<sup>-2</sup>), 0.1 % (10<sup>-3</sup>), and 0.01 % (10<sup>-4</sup>) v/v had already lost a significant number of culturable cells.
* **At 24 h post inoculation**, only the cultures seeded at the lowest concentrations maintained a high proportion of culturable cells.

The detailed results are found in Figure 2.

## Discussion

The results demonstrate that inoculum size strongly determines the quality of overnight cultures of *E. coli* in LB medium at 37 °C, a standard laboratory condition. After 10 h, all cultures appeared healthy, with culturable and total counts in close agreement. By 18 h, however, cultures seeded at 1 %, 0.1 %, and 0.01 % v/v had already lost a significant proportion of culturable cells. After 24 h, only the most lightly seeded cultures maintained high quality, while overseeded cultures contained large numbers of non-culturable cells.

These results highlight that standard laboratory workflows that rely on volumetric inoculation (for example ≥1 % v/v transfers) can easily compromise culture quality. To achieve a [high-quality culture](/mpd/encyclopedia/high-quality-culture.md) at the time of use, it is necessary to carefully determine the optimal inoculum concentration rather than blindly rely on fixed transfer volumes. For fast-growing bacteria under typical growth conditions, lower inoculation levels delay entry into stationary phase and yield healthier overnight cultures. While this study was performed with a fresh *E. coli* inoculum, the same principle applies more broadly: the optimal inoculum depends on strain, medium, and inoculum condition. Practical calibration with tools like BactoBox® ensures reproducibility and helps prevent overseeding from undermining culture quality.

## Want a how-to guide on identifying the right inoculum size with BactoBox®?

To obtain the results in Figure 2, you need extensive colony counting. You can avoid most plate counts by using a few BactoBox® measurements to get to similar conclusions about optimal seeding. The green caps in Figure 2 indicate the optimal cultures at specific HPIs.

For a practical guide, see [Optimize starter culture](/mpd/workflows/identify-optimal-inoculum-size.md).


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