Ultrastructure of coccoid viable but non‐culturable Vibrio cholerae

Morphology of viable but non-culturable Vibrio cholerae was monitored for 2 years by scanning and transmission electron microscopy. Morphological changes included very small coccoid forms, after extended incubation at 4°C and room temperature, and sequential transformation from curved rods to irregular (∼1 μm) rods to ∼0.8 μm coccoid cells and, ultimately, to tiny coccoid forms (0.07–0.4 μm). Irregular rod-shaped and coccoid cells were equally distributed in microcosms during the first 30–60 days of incubation at both temperatures, but only coccoid cells were observed after incubation for 60 days at 4°C. When V. cholerae O1 and O139, maintained for 30–60 days at both temperatures, were heated to 45°C for 60 s, after serial passage through 0.45 μm and 0.1 μm filters, and plating on Luria–Bertania (LB) agar, only cells larger than 1 μm yielded colonies on LB agar. Approximately 0.1% of heat-treated cultures were culturable. Cell division in the smallest coccoid cells was observed, yielding daughter cells of equal size, whereas other coccoid cells revealed bleb-like, cell wall evagination, followed by transfer of nuclear material. Coccoid cells of V. cholerae O1 and O139 incubated at 4°C for more than 1 year remained substrate responsive and antigenic.