Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers

Abstract

ABSTRACTAnaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions usingEscherichia coliDSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grownE. coliandClostridium bolteaeDSM 29485 to different doses of the toxin sodium arsenite. The growth ofE. coliandC. bolteaewas sensitive to arsenite doses of 1.3 μM and 0.4 μM, respectively. Complete inhibition of growth was achieved at 38 μM arsenite forC. bolteae, and 338 μM inE. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus.Importance statementWe present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications in which high-throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/toxin-microbial interactions.