Species-dependent colony size variation of environmental Enterococcus on sodium azide-containing selective agar

The gold standard for bacterial isolation is the selection of individual colonies from agar media. Selective media supplemented with chemical inhibitors are used to promote the growth of target bacteria while suppressing non-target organisms, particularly in environmental samples. Enterococcus, a genus comprising multiple species commonly found in bacteria-rich environments, can be isolated using agar supplemented with sodium azide. However, this approach generally enables identification only at the genus level, with limited discrimination among Enterococcus species. This study provides a detailed characterization of colony morphology in enterococci isolated from surface water using colony diameter as a discriminating parameter. At 0.4 g/L sodium azide, E. casseliflavus formed significantly smaller colonies (0.7 mm) than other Enterococcus species (>1.0 mm). Species forming colonies exceeding 1.0 mm were further characterized based on their extracellular polymeric substances (EPS). FTIR analysis of crude EPS (50-100 kDa) extracted by centrifugal ultrafiltration revealed similar spectral profiles among large-diameter E. hirae, E. lactis, and E. faecium, distinguishing them from E. faecalis. This study demonstrates that colony morphology on selective media can serve as a practical, culture-based approach for preliminary differentiation of selected Enterococcus species.