Overnight cultures

Explanation of how inoculum size affects overnight culture quality.

How you seed an overnight culture affects culture quality. Your culture may stay high-quality, or it may become dominated by stressed, non-culturable cells.

A high-quality culture has a high proportion of culturable bacterial cells.

A low-quality culture has a low proportion of culturable bacterial cells.

Introduction

It is common lab practice to use ≥1 % (v/v) seeding volume. This works for same-day expansion. But it 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^-2) at the high end.

• 0.00000001 % v/v (10^-11) 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 the subsequent flask.

Dilution series for overnight E. coli cultures. The initial dilution was 1:100 (10^-2) while remaining dilutions were 1:10.

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. 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. This is demonstrated by the superimposed BactoBox® (lavender) and CFU (yellow) data points and the CFU-to-cell ratio of ~1.

• At 18 h post inoculation, cultures seeded at 1 % (10^-2) and 0.1 % (10^-3) v/v had already lost a significant number of culturable cells. This is demonstrated by the divergence of the BactoBox® and CFU curve as well as the drop in CFU-to-cell ratio.

• At 24 h post inoculation, only the cultures seeded at the lowest concentrations (10^-9, 10^-10, and 10^-11) v/v maintained a high proportion of culturable cells.

The detailed results are shown in the below figure.

Too high seeding concentration may ruin overnight cultures. Baffled shake flasks with LB medium seeded with different concentrations of the same E. coli starter culture. The number in each box corresponds to the dilution factor. BactoBox® (lavender) and Compact Dry TC plate counts (yellow) are represented on the primary, logarithmic axis, and were done with duplicate repeats. CFU-to-cell ratio (green) is represented using the secondary axis. Secondary axis uses a linear representation for CFU-to-cell ratio. The labels represent the CFU-to-cell ratio for the green curve.

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 % and 0.1 % 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. In the gravest example, below ~⅓ of the cells were culturable after 24 h.

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 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.