Accelerate Bacterial Growth Measurement with Integrated Absorbance 96 Automate – Eppendorf epMotion® Automated Workflow

Key Highlights 

• Comparable levels of reproducibility, sensitivity, and linearity within the dynamic range were evident when comparing the performance of the Absorbance 96 Automate with that of another benchtop microplate reader.
• The Absorbance 96 Automate integrates seamlessly on-deck with the epMotion® liquid handling system to facilitate the automated measurement of bacterial growth.
• The precise liquid handling capabilities of the epMotion® system, combined with the rapid read-time proficiency of the Absorbance 96 Automate, collectively expedites the whole workflow compared to the manual approach. 

Introduction 

Monitoring the growth of bacteria in culture is crucial to understanding its growth kinetics and optimizing biomass and metabolite production (1). The most common method to quantify bacterial growth involves measurement of the optical density at 600 nm (OD600) utilizing either a microplate reader or photometer. Employing microplate readers for such measurements provides better temporal resolution and increased throughput (2). Automating microplate reading within a liquid handling system emerges as a crucial lab automation strategy to further enhance operational efficiency and high throughput in bacterial assays. The automated workflow solution mitigates labor-intensive procedures, including pipetting and cell seeding, enabling researchers to measure and analyze data from multiple plates with minimal user intervention expediently and precisely.
Moreover, specific challenges emerge when comparing benchtop microplate readers lacking integration with liquid handling systems. One such challenge involves the necessity for an additional gripper solution to facilitate the transfer of microplates from the liquid handling system to the proximal microplate reader. Thus, additional hardware-related complexities are introduced, which prove to be a cost-ineffective solution.  

In this collaborative study with Eppendorf, we demonstrate the performance of the Absorbance 96 Automate seamlessly integrated on-deck on the epMotion (5075t) liquid handling system for quantifying bacterial growth in an automated workflow. A comprehensive comparative analysis was performed between the Absorbance 96 Automate and another benchtop microplate reader to assess its performance. The results revealed comparable readouts, identical reproducibility, and performance sensitivity, thereby affirming the efficiency of the Absorbance 96 Automate in conducting automated OD600 measurements. Subsequently, a comparative evaluation of manual and automated laboratory workflows enabled by epMotion® revealed expedited steps in the automated workflow, resulting in a total time reduction for performing the whole assay. 

Automated Bacterial Growth Assay Workflow

Bacterial samples with varying dilution factors (2x, 4x, 8x, 16x, 32x, 64x, 128x) were prepared and dispensed in deep-well plates (DWP) in 8 replicates using epMotion® liquid handling system. Subsequently, the plates were cultured and incubated for 6 hours at 37 degrees, and afterward, bacterial aliquots were precisely pipetted into 96-well microplates (MTP) using the epMotion® system. The automatic measurement of absorbance at 595 nm was then performed by the Absorbance 96 Automate, positioned on the deck of the epMotion® workstation. 

Results 

Absorbance 96 Automate and GloMax® Discover Exhibit Comparable OD600 Readouts and Performance Values

To validate the performance of the Absorbance 96 Automate, a comparative analysis was conducted with the GloMax® Discover microplate reader from Promega. Bacterial samples with varying dilution factors were measured by recording absorbance values at 595 nm using Absorbance 96 Automate or at 600 nm using GloMax® Discover microplate reader. Similar OD values and a significant correlation (R² = 0.9991) were observed between both instruments (Fig. 1).

The results affirm that the Absorbance 96 Automate exhibits identical reproducibility, sensitivity, and linearity within the evaluated dynamic range compared to the GloMax® Discover microplate reader. Notably, the Absorbance 96 Automate demonstrated a reduced readout time of 5 seconds compared to the GloMax® Discover microplate reader with 58 seconds, underscoring its efficiency in rapid data acquisition. 

Automated OD600 Measurements Enhance Efficiency in Bacterial Growth Analysis

Subsequently, a comparative analysis was conducted to assess bacterial growth using manual and automated workflows employing the epMotion® — Absorbance 96 Automate workflow solution. The outcomes revealed a substantial correlation between the two methodologies (Fig. 2), indicating similar efficient performance of the automated workflow to the manual approach.

In contrast to the manual workflow, the total workflow analysis time decreased significantly from 19 to 14.5 minutes in the automated workflow, facilitating direct plate reading without requiring user intervention.

Summary 

Integrating Absorbance 96 Automate onto the epMotion deck offers a notably convenient and expeditious approach for assessing bacterial growth in a 96-well plate. Comparative analysis results between Absorbance 96 Automate and GloMax® Discover validate the robust performance of Absorbance 96 Automate for performing OD600 measurements, showcasing identical reproducibility, sensitivity, and linearity. Furthermore, the observed time-saving advantage in the automated workflow compared to the manual workflow emphasizes the reliability and robustness of the epMotion®-Absorbance 96 Automate synergy as an effective and expeditious solution for automated bacterial growth measurement across diverse assays.

References

1. Loutfi H. et al. Real-time monitoring of bacterial growth kinetics in suspensions using laser speckle imaging, Scientific Reports 2020 10:1, 10(1).
2. Krishnamurthi VR. et al. A new analysis method for evaluating bacterial growth with microplate readers, PLoS ONE 2021, 16.