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

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.