Enhanced Reproducibility and Precision of High-Throughput Quantification of Bacterial Growth Data Using a Microplate Reader

Summary

Here, a high-throughput protocol is presented to measure growth data, including growth curves, growth rate, and maximum growth rate. The protocol was verified and validated using two biofilm-producing bacteria. The results and approach applied in this study can be expanded to other high-throughput protocols using microplate readers.

Abstract

This study aimed to develop a repeatable, reliable, high-throughput protocol to monitor bacterial growth in 96-well plates and analyze the maximum growth rate. The growth curves and maximum growth rates of two bacterial species were determined. Issues including (i) lid condensation, (ii) pathlength correction, (iii) inoculation size, (iv) sampling time interval, and (v) spatial bias were investigated. The repeatability of the protocol was assessed with three independent technical replications, with a standard deviation of 0.03 between the runs. The maximum growth rates of Bacillus mycoides and Paenibacillus tundrae were determined to be (mean ± SD) 0.99 h⁻¹ ±  0.03 h⁻¹ and 0.85 h⁻¹ ± 0.025 h⁻¹, respectively. These bacteria are more challenging to monitor optically due to their affinity to clump together. This study demonstrates the critical importance of inoculation size, path length correction, lid warming, sampling time intervals, and well-plate spatial bias to obtain reliable, accurate, and reproducible data on microplate readers. The developed protocol and its verification steps can be expanded to other methods using microplate readers and high-throughput protocols, reducing the researchers' innate errors and material costs.

Introduction

Developing interest in multi-omics manipulation, including mechanism and metabolic studies of bacteria, emphasizes the importance of high-throughput and automated methods such as recording growth data^1,2. Growth data comprising kinetic parameters, such as maximum growth rates, can help characterize bacterial responses to different physical, chemical, and antibacterial conditions. Growth rate data are a standard response variable utilized to uncover potential genotype-phenotypes linkages¹ or indicate the microbial safety and shelf life of food produce

Protocol

NOTE: All steps in this protocol must be followed in sterile conditions (i.e., between two flames or a biosafety cabinet). All materials and tools are autoclaved for 20 min. See the Table of Materials for details about all materials, equipment, and software used in this protocol. Gloved hands are disinfected, kept wet with hand disinfectant or 70% alcohol solution for at least 1 min, and not removed from the safety cabinet afterward. Otherwise, the disinfecting procedure must be repeated before introduci.......

Discussion

Microplate readers allow for obtaining consistent and repeatable growth rates. This technology minimizes human error and enables high-throughput sampling. The small amount of culture required per sample makes this approach an attractive, low-cost alternative to cell counts using flasks or test tubes. Microplate readers allow a large sample size, increasing the statistical power and subsequently facilitating reliable growth rate calculations while keeping costs and labor low.

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Materials

Name	Company	Catalog Number	Comments
Centrifuge	Eppendorf	5810 R
Centrifuge tubes - 15 mL	ThermoFisher- Scientific	339650	Sterile
Centriguge tubes - 50 mL	ThermoFisher- Scientific	339652	Sterile
Disposable inoculating loop , 10 µL	Cole-Parmer	UZ-06231-08	Sterile
Erlenmeyer flasks - 250 mL	Cole-Parmer	UZ-34502-59	Glass
Isopropanol	ThermoFisher- Scientific	396982500	≥99.0
Phosphate Buffer Saline	Sigma-Aldrich	P4417
Pipett tips 1,000 µL	ThermoFisher- Scientific	UZ-25001-76
Pipett tips 10 mL	ThermoFisher- Scientific	UZ-25001-83
Pipett tips 200 µL	ThermoFisher- Scientific	UZ-25001-85
Pipett tips 5 mL	ThermoFisher- Scientific	UZ-25001-80
Pipettor 1,000 µL	Cole-Parmer	UZ-07909-11
Pipettor 10 mL	Cole-Parmer	UZ-07909-15
Pipettor 200 µL	Cole-Parmer	UZ-07909-09
Pipettor 5 mL	Cole-Parmer	UZ-07859-30
Tryptic Soy Broth	Millipore	22091	Suitable for microbiology