The viable but non-culturable state in Vibrio cholerae O1 and O139

Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4°C, a condition that induces the viable but non-culturable (VBNC) state. Cells were fixed at different times during entry into the VBNC state. When no growth was detectable in either solid or liquid media, the ultra structure of these cells was examined, using both transmission and scanning electron microscopy. The cells of both strains became smaller in size and changed from curved rods to short rods to ovoid and then coccoid morphology. Results of transmission electron microscopy revealed the central region of the cells to be compressed and surrounded by denser cytoplasm when the time of incubation was long, i.e., 6 months to 1 year. VBNC cells grown in 0.5% yeast extract and heat shocked at 45°C for 1 min continued to demonstrate cell division. Coccoid VBNC showed not only cell division but also release very small coccoid cells 0.2--0.4 mu in size. These very small coccoid cells demonstrated cell division when viewed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The viability of these very small coccoid cells was examined. The number of the very small coccoid VBNC cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacteria Viability kit), with 75 to 90% of the cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. Coccoid cells and the very small coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with fluorescent monoclonal antibody. Viability of the very small VBNC cells was further established by measuring their susceptibility to chorine, copper sulfate, zinc sulfate, and formaldehyde.;VBNC cells were resuscitated by exposure to heat at 45°C for 60 sec and 65°C for 30 sec. Approximately 1% of the VBNC cells were recovered and able to form colonies on LB agar. When heat activation was applied to VBNC cells subjected to size exclusion, growth on LB agar was observed only of cells larger than 1 mu under similar activation conditions. VBNC cells smaller than 1 mu demonstrated cell division when examined by TEM and SEM, although these cells failed to form colonies on LB agar.;Since retention of cell membrane integrity is a determining characteristic of viable cells, chromosomal DNA was extracted from the very small coccoid VBNC cells in microcosms that had been maintained for two months and for one year. Conservation of the cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of the very small coccoid VBNC cells was demonstrated using PCR and employing specific primers.