Isolation of Extracellular Vesicles From Gram‐Negative Bacteria: A Solid Medium‐Based Method Analysis

ABSTRACT Extracellular vesicles (EVs) produced by gram‐negative bacteria have gained increased scientific interest over the last years due to a growing understanding of their roles in pathogenesis and biotechnological potential. However, isolating such nanoparticles is technically challenging. Here, we evaluated a rapid and reproducible protocol for EV isolation from bacteria grown on solid medium, specifically analyzing Gram‐negative species within the genera Acinetobacter , Aeromonas , Escherichia , Enterobacter , Klebsiella , and Pseudomonas . In addition to avoiding large culture volumes, the method was reproducible, relatively fast, compatible with multiple downstream analyses, including flow cytometry, TEM, and NTA, while yielding strain‐specific nanoparticle profiles with acceptable purity. Our results also show that Pseudomonas and Aeromonas strains were the highest and lowest EV producers, respectively. Although the method frequently co‐isolated filaments, clean isolates were also obtained. Comparison of EV isolation from cells grown on solid medium with those grown in liquid cultures showed that solid growth consistently yielded more nanoparticles. The methodology represents a valuable approach for bacterial EV isolation and may serve as an important initial step in EV research workflow.