Development of a high‐throughput method for examining bacterial supernatant pH using ratiometric UV‐VIS spectrophotometry

Abstract

Background Bacterial supernatant pH measurements are highly relevant to downstream applications, such as organoid or cell line treatment, mass spectrometry, and metabolic analysis. Although high-throughput systems have been implemented for examining bacterial growth, high-throughput systems for measuring bacterial supernatant pH are lacking. The traditional method for measuring the solution pH of bacterial supernatants uses pH meters and electrodes. However, this method is not compatible with high throughput approaches which employ microplate readers. While fluorescent dyes can be used to measure solution pH, bacterial media is highly auto-fluorescent, making this system incompatible for microbial analysis. To overcome these limitations, we have developed a simple, high-throughput assay to measure bacterial supernatant pH using ratiometric UV-VIS spectrophotometry and phenol red, the pH indicator dye contained in the medium DMEM. Phenol red exhibits a gradual transition from yellow to fuchsia from acid to basic solution pH, respectively. The aim of this study was to develop a high-throughput and accessible method to monitor pH changes in multiple samples using a standard microplate reader. Methods & Results As a proof of concept, we prepared a series of solutions with pHs that ranged from 3 to 10 in DMEM and DMEM containing a fully defined bacterial media, termed ZMBI, at a ratio 3:1. UV-VIS spectral analysis of phenol red from 350-750 nm confirmed the reported wavelengths for the isosbestic point - a specific wavelength at which the total absorbance of an indicator dye remains consistent – and the wavelength of maximum absorption (λmax) to be 470 and 560 nm, respectively. Calibration curves generated by the ratio of these two wavelengths (560 nm/470 nm) revealed a sigmodal curve, which could be fit by a four-parameter logistics curve. To test the ability of this system to correctly calculate pH, commensal gut microbes Lactobacillus reuteri and Escherichia coli were grownin ZMBI overnight anaerobically and aerobically, respectively. A comparison between standard pH electrode analysis and our newly developed wavelength analysis revealed similar calculated pHs. To generate a high-throughput system, L. reuteri and E. coli were grown in 1 mL of ZMBI in DeepWell microplates overnight. To examine bacterial growth, 100 µL of culture was transferred to flat bottomed 96-well microplates and optical density was measured at OD600nm. For pH analysis, a 200-µL volume of culture was transferred to round bottomed 96-well plates, centrifuged and cell-free supernatant was blended with ZMBI at a 3:1 ratio. Back calculated solution pH values using the sigmodal calibration curve yielded similar results to the pH electrode values. Conclusions We have successfully employed DMEM's phenol-red dye, which is well suited for a colorimetric assay, to study bacterial supernatant pH. This method allows for rapid and accurate pH measurement which can be easily be applied to multiple organisms and adapted to several downstream applications.