A rapid, reliable, and cost-effective method for monitoring Mycoplasma spp. growth in real-time using Resazurin fluorescence

Mycoplasmas are small, slow-growing bacteria that do not produce visible turbidity in broth. Monitoring the growth of these fastidious organisms requires the manual sampling of cultures over several days, followed by cumbersome enumeration methods with long incubation periods or complex assays unable to differentiate live and dead cells. Here, a simple, automated assay was developed to measure Mycoplasma growth by quantifying the reduction of resazurin, a non-toxic dye, into a fluorescent product by live organisms. Mycoplasma species were cultivated in broth containing 2.5 mg/L resazurin. Fluorescence (520 nm excitation, 555 nm emission) was recorded every 5 min for 24 h using a qPCR thermocycler set at 37 °C, to capture the logarithmic and plateau phases. Growth curves and generation times obtained from fluorescence readings were highly similar to those calculated from the Most Probable Number (MPN) titres analysis of broth cultures sampled every 2 h over the same timeframe. Additionally, the resazurin assay could rapidly differentiate temperature-sensitive mutants from wild-type strains, by comparing their maximal growth rates in permissive (33 °C) and non-permissive (39 °C) conditions. While the MPN titration protocol required tedious liquid handling and weeks-long incubation to produce interpretable data, the resazurin assay delivered results in less than 24 h. Unlike the MPN method, which relies on pH changes, the resazurin assay could be used with non-acidifying Mycoplasmas. In conclusion, resazurin-based fluorescence monitoring provides a practical and accurate solution to quantify Mycoplasma growth, with strong potential for diagnostic and research applications.