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) 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.

Experimental growth curve for Klebsiella aerogenes. BactoBox® total cell counts are shown in lavender, while colony-forming units (CFU/mL) are shown in yellow. Total cell counts and CFU/mL are superimposed up to about 12 h (grey box), which means that practically all cells are culturable. When the culture enters death stage, a high proportion of cells become non-culturable.

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 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. To account for CFU variability, we considered 80–125 % as high-quality.

A starter culture of E. coli ATCC 8739 was used to seed 10 shake flasks in a dilution series ranging from 10^-2 to 10^-11. Each dilution was measured with BactoBox® and CFU at 10, 18, and 24 h post inoculation.

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^-2), 0.1 % (10^-3), and 0.01 % (10^-4) 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.

Figure 2: Comparison of BactoBox® cell concentrations (lavender) and CFU concentrations (yellow), analyzed at 10 h, 18 h, and 24 h post inoculation (HPI). In the left panel, bars represent concentrations from cultures seeded at 1 % (10^-2) down to 10^-11 (v/v) using a 1:10 dilution series in LB medium. The right panel shows the proportion of culturable cells (CFU) relative to the total cells (BactoBox®). Flasks with green caps indicate the highest quality culture at the designated incubation time.

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 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 inoculation ratio 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.

Optimize starter culture gives you a simple workflow for finding the right inoculation ratio thereby shortening the lag phase duration for the next step in your bioprocess.