Overnight cultures

Explanation of how inoculum size affects overnight culture quality.

The way an overnight culture is seeded has a profound influence on whether the final population will be that of High-quality culture, or whether it will be dominated by stressed and non-culturable cells.

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

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

Introduction

It is common lab practice to use ≥1% (v/v) seeding volume when inoculating cultures. While this may be great for fast, same-day culture expansion, it can easily ruin overnight cultures: the cells quickly reach stationary stage and enter death phase.

The growth curve below illustrates the growth pattern of the fast-growing species, Klebsiella aerogenes. The exponential phase completes in approximately 6 hours, after which the culture enters the deceleration, stationary, and death phases. In effect the culture will be of relatively low quality after the overnight incubation.

Fortunately, overseeding is easy to prevent: Simply use a lower initial seeding concentration to prolong the exponential growth phase before nutrient depletion. Below is a simple data example illustrating this practice.

Experimental growth curve for Klebsiella aerogenes. BactoBox total cell counts are shown in lavender, while colony-forming units (CFUs) are shown in yellow. Total cell counts and CFUs are superimposed up to ~12 hours (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 in which a stationary stage, liquid culture of E. coli ATCC 8739 was used to seed 10 shake flasks using different volumes. The highest seeding volume was 1% v/v (10^-2), and the lowest seeding volume was 0.00000001% v/v (10^-11). The latter was achieved through a dilution series where 5 mL was transferred from the preceding flask to 45 mL fresh medium in a new flask. All 10 shake flasks were incubated at 37 °C, 150 rpm and analyzed at 10h, 18h and 24h. Culture quality was assessed by comparing the concentration of culturable cells (via plate counts) to the total cell concentration (via BactoBox® BacTotal-2024-10 using ECC-factor of 1.27). A ratio close to 1 (100%) indicates a High-quality culture. To account for the inherent variability of CFU determinations, cultures with ratios between 80% and 125% were considered 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 CFUs at 10, 18, and 24 hours post inoculation.

Results

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

• At 10 hours post inoculation, all cultures were healthy: culturable cell counts and total cell counts matched closely.

• At 18 hours 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 hours 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) at analyzed at 10h, 18h, 24h 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 hours, all cultures appeared healthy, with culturable and total counts in close agreement. By 18 hours, however, cultures seeded at 1%, 0.1%, and 0.01% v/v had already lost a significant proportion of culturable cells. After 24 hours, 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 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, extensive colony-counting is required. However, you can avoid the tedious plate counts as just a few BactoBox measurements can help you reach similar conclusions about optimal seeding concentrations. Notice the green caps in Figure 2; they indicate the optimal cultures at specific HPIs. For guidance on determining the appropriate inoculum size, check out our simple how-to guide here: Get high-quality overnight cultures from cryo stocks.