Physiological, Microscopic, and Molecular Approaches to Characterize Methane-Oxidizing Methylosinus sp. Strain C49: Culture Revival, Fluorescence Imaging Optimization, and Purity Assessment

This study presents a multimodal characterization of Methylosinus sp. strain C49 (MC49), a Type II methanotroph, to establish a reproducible workflow for methane mitigation and bioplastic polyhydroxyalkanoate (PHA) production. Cryopreserved cultures exhibited measurable growth by day 10, peaking at OD₆₀₀ 0.357 by day 14. Fluorescence microscopy identified SYBR Green I combined with SlowFade™ Diamond as optimal for cell visualization, while PCR-RFLP confirmed genetic purity across subcultures, storage methods, and environmental conditions, with minor changes after prolonged starvation. This integrated approach provides a reliable framework for methanotroph-based biotechnological applications and underscores critical considerations for culture maintenance, imaging, and molecular validation.